Blocking CD40-TRAF6 Interactions by Small-Molecule Inhibitor 6860766 Ameliorates the Complications of Diet-Induced Obesity in Mice

ORIGINAL ARTICLE Blocking CD40-TRAF6 interactions by small-molecule inhibitor 6860766 ameliorates the complications of diet-induced obesity in mice

SM van den Berg1,7, TTP Seijkens1,7, PJH Kusters1, B Zarzycka2, L Beckers1, M den Toom1, MJJ Gijbels1,3,4, A Chatzigeorgiou5, C Weber2,6, MPJ de Winther1, T Chavakis5, GAF Nicolaes2 and E Lutgens1,6

BACKGROUND: Immune processes contribute to the development of obesity and its complications, such as insulin resistance, type 2 diabetes mellitus and cardiovascular disease. Approaches that target the inflammatory response are promising therapeutic strategies for obesity. In this context, we recently demonstrated that the interaction between the costimulatory protein CD40 and its downstream adaptor protein tumor necrosis factor receptor-associated factor 6 (TRAF6) promotes adipose tissue inflammation, insulin resistance and hepatic steatosis in mice in the course of diet-induced obesity (DIO). METHODS: Here we evaluated the effects of a small-molecule inhibitor (SMI) of the CD40-TRAF6 interaction, SMI 6860766, on the development of obesity and its complications in mice that were subjected to DIO. RESULTS: Treatment with SMI 6860766 did not result in differences in weight gain, but improved glucose tolerance. Moreover, SMI 6860766 treatment reduced the amount of CD45+ leucocytes in the epididymal adipose tissue by 69%. Especially, the number of adipose tissue CD4+ and CD8+ T cells, as well as macrophages, was significantly decreased. CONCLUSIONS: Our results indicate that small-molecule-mediated inhibition of the CD40-TRAF6 interaction is a promising therapeutic strategy for the treatment of metabolic complications of obesity by improving glucose tolerance, by reducing the accumulation of immune cells to the adipose tissue and by skewing of the immune response towards a more anti-inflammatory profile. International Journal of Obesity (2015) 39, 782–790; doi:10.1038/ijo.2014.198

INTRODUCTION cardiovascular disease.5–10 Strategies that modulate the inflam- Obesity and its associated conditions, including insulin resistance, matory response are therefore promising therapeutic modalities type 2 diabetes mellitus and cardiovascular diseases, affect more for obesity and its complications. than 1000 million people worldwide, a number that is increasing The costimulatory dyad CD40 and its ligand CD40L (CD154) and is expected to do so for the next decades.1,2 Chronic caloric have a well-known role in immune cell activation and fl 11 excess may shorten healthy lifespan of humans by 5 − 20 years, in ammation. After binding of CD40L, CD40 recruits adaptor which results in a tremendous socioeconomic burden.1 Hence, the proteins, the tumor necrosis factor receptor-associated factors (TRAFs).11 The intracellular domain of CD40 contains two binding development of novel therapeutic strategies for obesity and its sites for these proteins: a proximal site that binds TRAF2/3/5 and a related disorders is a public health priority. distal site that binds TRAF6.11 After binding of the TRAF proteins Over the past decades, chronic inflammation has been to CD40, signal transduction is initiated, which eventually results identified as the pathological substrate of obesity and its 11 1,3,4 in the expression of inflammatory mediators. Mice with a complications. Elevated plasma levels of cytokines, such as genetic deficiency in the CD40-TRAF6 interaction, but not in the tumor necrosis factor and interleukin-6 (IL-6), characterize obese CD40-TRAF2/3/5 interaction, are protected against the develop- subjects and show a positive correlation with the extent of 5–9 ment of neointima formation and atherosclerosis, both exponents metabolic dysfunction. Obese adipose tissue (AT) exhibits of an ongoing inflammatory condition of the arterial wall.12,13 hallmarks of an ongoing inflammatory response, such as immune Accumulating evidence indicates that the CD40-CD40L dyad cell infiltration and activation, as well as the presence of has an important role in obesity.6,14–17 For example, plasma levels proinflammatory cytokines and adipokines, including tumor of soluble CD40L correlate with the body mass index in obese – necrosis factor, IL-6 and leptin.5 9 Moreover, chronic inflammation subjects and decrease after bariatric surgery.14 CD40-CD40L- has a pivotal role in the development of obesity-associated mediated interactions between adipocytes and AT immune cells, diseases, such as hepatic steatosis, type 2 diabetes mellitus and including T cells and macrophages, promote the expression of

1Department of Medical Biochemistry, Subdivision of Experimental Vascular Biology, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; 2Department of Biochemistry, University of Maastricht, Maastricht, The Netherlands; 3Department of Pathology, Maastricht University, Maastricht, The Netherlands; 4Department of Molecular Genetics, Maastricht University, Maastricht,The Netherlands; 5Department of Clinical Pathobiochemistry and Institute for Clinical Chemistry and Laboratory Medicine, Medical Faculty, Technische Universität Dresden, Dresden, Germany and 6Institute for Cardiovascular Prevention (IPEK), Ludwig Maximilian’s University, Munich, Germany. Correspondence: Professor Dr E Lutgens, Department of Medical Biochemistry, Subdivision of Experimental Vascular Biology, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, Amsterdam 1105 AZ, The Netherlands. E-mail: [email protected] 7These authors contributed equally to this work. Received 4 September 2014; revised 10 October 2014; accepted 27 October 2014; accepted article preview online 13 November 2014; advance online publication, 9 December 2014 SMI-mediated CD40-TRAF6 inhibition in obesity SM van den Berg et al 783 proinflammatory cytokines and chemokines, which increase the mice were treated daily with SMI 6860766 (10 μmol kg − 1)(n = 12) or recruitment of other inflammatory cells to the AT.15,16 Ligation of vehicle (0.05% Tween-80, 5% dimethyl sulphoxide in phosphate-buffered CD40 on adipocytes promotes insulin resistance by decreasing saline)19 (n = 12) for 6 weeks. Mice had ad libitum access to food and water insulin receptor substrate-1 and glucose transporter type 4 and were maintained under a 12 h light–dark cycle. Food intake and body expression.15,16 Genetic deficiency or antibody-mediated inhibition weight were measured weekly. After the experimental procedure, mice of CD40L reduced the inflammatory and metabolic complications of were fasted overnight and subsequently killed. Blood was collected and fl diet-induced obesity (DIO).15,16 In contrast, deficiency of CD40 organs were dissected and processed for ow cytometric and histological aggravates the complications of DIO.18–20 To understand these analysis. All experimental procedures were approved by the Animal Experimentation Ethics Committee of the University of Amsterdam. opposing effects of CD40L and CD40, we previously investigated the role of different downstream adaptor proteins of the CD40-CD40L pathway. We found that deficiency of CD40-TRAF2/3/5 interactions Haematology and biochemical measurements increased the metabolic and inflammatory complications of DIO, Blood was obtained by cardiac puncture with EDTA-filled syringes. whereas deficiency of CD40-TRAF6 interactions rather improved AT Haematological analysis was performed on a ScilVet abc plus+ (ScilVet, inflammation and insulin resistance in DIO mice. Based on these Oostelbeers, The Netherlands). data, we developed a small-molecule inhibitor (SMI) against CD40- TRAF6 interactions, and we could show that treatment with SMI Insulin assay and calculation of HOMA-IR 6877002 improved insulin resistance, hepatic steatosis and M1 Fasting insulin was measured in plasma by enzyme-linked immunoabsor- macrophage accumulation into the epididymal AT (EpAT), when bent assay (Mercodia, Uppsala, Sweden) following the manufacturer's treatment was initiated 6 weeks after the beginning of the diet.19 protocol. Glucose levels were measured from whole blood upon killing Thus, inhibition of the CD40-TRAF6 interaction is a promising using a glucometer (Bayercontour, Basel, Switzerland). The homeostasis therapeutic strategy for obesity. model assessment of insulin resistance (HOMA-IR) was calculated using the − 1 Here we performed a more thorough analysis of the effects of following formula: HOMA-IR = fasting glucose (mmol l ) × fasting insulin − 1 SMI-mediated CD40-TRAF6 inhibition in DIO. We investigated the (m U l )/22.5. effects of SMI 6860766, an analogue of 6877002 with a lower IC50 (half-maximal inhibitory concentration),19 in DIO. In addition, we Glucose tolerance test here administered the CD40-TRAF6 inhibitor after 12 weeks of One week before and 3 and 6 weeks after the initiation of SMI 6860766 or high-fat diet (HFD), when obesity was much more progressed, and vehicle treatment, a glucose tolerance test was performed. Twelve-hour continued diet and treatment until week 18. Our data suggest that fasted mice were injected intraperitoneally with glucose (1 mg g − 1; Sigma- SMI-mediated CD40-TRAF6 inhibition is a promising novel Aldrich, Zwijndrecht, The Netherlands). Glucose levels were measured from therapeutic approach in DIO-related metabolic dysregulation. whole blood using a glucometer (Bayercontour, Basel, Switzerland) at times indicated in the figures.

MATERIALS AND METHODS Real-time PCR TRAF6 C-domain expression, purification and binding analysis Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) and His-tagged TRAF6 C-domain (residues 346–504) was expressed in reverse transcribed with an iScript cDNA Synthesis Kit (Bio-Rad, Escherichia coli using the pET21d expression vector (Novagen, Billerica, Veenendaal, The Netherlands). Quantitative PCR was performed using a MA, USA). Protein was purified by affinity chromatography, followed by gel SYBR Green PCR Kit (Applied Biosystems, Leusden, The Netherlands) on a filtration in running buffer (25 mM Tris, 200 mM NaCl and 0.5 mM TCEP). The ViiA7 Real-Time PCR System (Applied Biosystems). The result is expressed direct binding between the TRAF6 C-domain and SMI 6860766 was as relative to the control group, which was assigned a value of 1. measured by surface plasmon resonance (Biacore T200; GE Healthcare, Eindhoven, The Netherlands). TRAF6 C-domain was immobilized on Sensor Chip CM5 using the amine coupling method. This reached a density of Flow cytometry ~ 12 000 and 7500 resonance units (RU). SMI 6860766 was dissolved in EpAT and subcutaneous AT (ScAT) were removed, rinsed in phosphate- phosphate-buffered saline buffer containing 5% (v v− 1) dimethyl sulph- buffered saline and minced into small pieces. Tissues were digested in a oxide. All measurements were carried out at 25 °C and with a flow rate of collagenase mixture (DMEM-20 mM HEPES, collagenase I and collagenase − 50 μl min 1 in surface plasmon resonance running buffer (phosphate- XI; Sigma-Aldrich) for 45 min at 37 °C. The digested samples were passed buffered saline, 0.05% Tween-20, 5% dimethyl sulphoxide, pH = 7.4). through a 70-μm nylon mesh (BD Biosciences, Breda, The Netherlands). Sensorgrams were corrected by subtracting the initial level of surface The suspension was centrifuged at 1250 r.p.m. for 6 min and the pelleted plasmon resonance signal before injection of the SMI or the TRAF6 stromal vascular fraction was resuspended in FACS buffer. C-domain. Data were analysed using the BIA Evaluation software (GE Erythrocytes in blood were removed by incubation with hypotonic lysis Healthcare). Equilibrium dissociation constants (Kd) were determined from buffer (8.4 g of NH4Cl and 0.84 g of NaHCO3 per litre of distilled water). To a model of the steady-state affinity (three independent runs were prevent nonspecific binding of antibodies to the Fc receptor, all cell averaged). suspensions were incubated with a CD16/32 antibody before labelling. CD3, CD8, CD25, FoxP3, F4/80, CD11b, CD11c, Gr-1 (eBioscience, San Diego, In vitro macrophage culture CA, USA), CD4, Ly6G (BD Biosciences) and CD206 (BioLegend, San Diego, Bone marrow (BM) cells were isolated from CD40+/+ and CD40 −/− mice (C57Bl/ CA, USA) antibodies were incubated with the indicated tissues. Staining fl 6background),21 as well as mice containing site-directed mutagenesis for the was analysed by ow cytometry (FACSCanto II; BD Biosciences) and by respective CD40-TRAF-binding domains (CD40-Twt, CD40-TRAF2/3/5−/− and using the FlowJo software version 7.6.5. (Tree Star, Ashland, OR, USA). CD40-TRAF6−/ −)22 and cultured in RPMI supplemented with 15% L929 conditioned medium to generate BM-derived macrophages. BM-derived Histology macrophages were activated with an agonistic CD40 antibody cocktail FGK45 − Tissues were collected, fixed in 4% paraformaldehyde and embedded in and 5D12 (both 25 μgml 1; Bioceros BV, Utrecht, The Netherlands) overnight, paraffin. Liver steatosis and inflammation were graded on 4-μM-thick incubated with SMI 6860766 for 1 h and frozen for real-time PCR analysis. haematoxylin-and eosin-stained sections. Immunohistochemistry on liver, EpAT and ScAT was performed for CD45 (BD) and on EpAT for CD68 (Bio- Mice Rad). Five micrometre frozen sections of the liver were stained with Oil Red Male C57Bl/6 mice (n = 24) were purchased from Charles River (Leiden, The O (Sigma-Aldrich). Organs were analysed by haematoxylin and eosin Netherlands) and maintained at the animal facility of the University of staining. Morphometric analyses were performed using the Las4.1 software Amsterdam (Amsterdam, The Netherlands). Mice received an HFD (35% (Leica, Rijswijk, The Netherlands) and the ImageJ software (Bethesda, MD, kcal carbohydrate, 45% kcal fat, 20% kcal protein; Special Diets Services, USA). Analyses were performed by an observer who was blinded for the Witham, UK) for 18 weeks from the age of 7 weeks. After 12 weeks of HFD, experimental conditions.

© 2015 Macmillan Publishers Limited International Journal of Obesity (2015) 782 – 790 SMI-mediated CD40-TRAF6 inhibition in obesity SM van den Berg et al 784 Statistical analysis antibodies 5D12 and FGK45. When stimulating these macro- +/+ − / − Results are presented as mean ± s.e.m. Data were analysed by a Student’s phages overnight, CD40 , CD40-Twt and CD40-TRAF2/3/5 t-test or χ2 test using the GraphPad Prism 5.0 software (GraphPad Software macrophages display a high expression of CCL2, whereas CCL2 − − − − Inc., La Jolla, CA, USA). P-values o0.05 were considered significant. was significantly reduced in CD40 / and CD40-TRAF6 / macrophages, proving that CD40-TRAF6 interactions are respon- sible for the decrease in CCL2 levels. When adding SMI 6860766, RESULTS together with the CD40-activating antibody, CCL2 levels were also Characterization of SMI 6860766 reduced in CD40+/+, CD40-Twt and CD40-TRAF2/3/5− / − macro- The CD40-TRAF6 SMI 6860766 is specifically modelled for the phages, suggesting that the CD40-TRAF6 inhibiting SMI 6860766 is CD40-TRAF6 binding site on the TRAF6 molecule; the molecular specific for CD40-TRAF6 interactions (Figure 1d). structure of SMI 6860766 is shown in Figure 1a. Biacore analysis shows that SMI 6860766 binds well to the part of the TRAF6 Small-molecule treatment did not result in side effects molecule in which the CD40 binding site is present (Figure 1b). In We previously developed small-molecule inhibitors of the CD40- vitro, SMI 6860766 dose-dependently suppressed CD40-induced TRAF6 interaction,19 and found no side effects for SMI 6877002. In gene expression of IL-1β and IL-6 cytokines in BM-derived the present study, we administered SMI 6860766, an analogue of fi macrophages (Figure 1c). Further functional speci city can be 6877002 with a lower IC50, in a more progressed model of DIO deducted from the following experiment: BM-derived macro- where treatment with the CD40-TRAF6 inhibitor was started after − − − − phages from CD40+/+, CD40 / , CD40-Twt, CD40-TRAF2/3/5 / 12 weeks of HFD and continued until week 18 of HFD. We did not − − and CD40-TRAF6 / mice (mice that contain a site-directed observe any side effects. Daily treatment with SMI 6860766 for the mutagenesis in the CD40 gene for the respective TRAF-binding past 6 weeks of 18 weeks of HFD did not result in any changes domain on CD40) were activated with the CD40 agonistic in peripheral blood leucocyte counts or cell composition

6860766 100 µm 20 50 µm 18 16 25 µm 14 H 125 µm N 12 10 63 µm 8 µ 0 6 3.1 m Response (RU) CI Br 4 1.6 µm 2 0 40 60 80 100 120 140 160 180 200 Time (s) IL-6 IL-1β

CCL2

Figure 1. Characterization of SMI 6860766. (a) Molecular structure of SMI 6860766. (b) Surface plasmon resonance sensogram of SMI 6860766, which conﬁrmed the direct binding of SMI 6860766 to immobilized TRAF6 C-domain. Data represent the average of three independent experiments. (c) Dose-dependent inhibition of IL-6 and IL-1β gene expression in FGK45 (agonistic CD40 antibody) stimulated BM-derived macrophages. (d) CCL2 gene expression in BM-derived macrophages of the respective genotypes stimulated with the CD40 agonistic antibodies FGK45 and 5D12 (n = 6 per group; **Po0.01 and ***Po0.001).

EpAT ScAT

EpAT

CTRLTRL 68607666860766

Figure 2. Daily treatment with SMI 6860766 during the past 6 weeks of 18 weeks HFD reduced adipocyte size and improved glucose sensitivity in mice. (a) Treatment with SMI 6860766 did not result in differences in body weight gain. (b) Gene expression of CD40 showing a higher expression of CD40 in EpAT compared with ScAT. (c) Adipocyte size of EpAT and ScAT showing a 15.3% decrease in epididymal adipocyte size of SMI 6860766-treated mice as indicated by an increase in adipocyte numbers per ﬁeld (n = 12 per group). (d) Haematoxylin and eosin staining illustrating that epididymal adipocyte size was decreased in SMI 6860766-treated mice compared with vehicle. (e) Glucose tolerance test (GTT) of mice fed an HFD for 15 weeks with 3 weeks of SMI 6860766 treatment and GTT of mice fed an HFD for 18 weeks with 6 weeks of treatment showing improved glucose sensitivity in mice treated with SMI 6860766 compared with vehicle (n = 6 per group; *Po0.05, **Po0.01 and ***Po0.001).

(Supplementary Figures 1a–f). Macroscopic and histopathological body weight gain or food intake between the experimental analysis revealed no abnormalities of the SMI treatment in more groups (Figure 2a and Table 1). SMI 6860766 treatment did not than 20 organs analysed, which included spleen, colon, small affect EpAT, ScAT or BAT weights (Table 1). Because SMI 6860766 intestine, stomach, kidney, lung, heart and muscle. binds to CD40-TRAF6, we conﬁrmed the presence of CD40 in the ATs and found an 11-fold higher expression of CD40 in EpAT Alterations in adipocyte size in the AT compared with ScAT in the control group, indicating that the SMI During the course of DIO, both groups showed a signiﬁcant gain can exert larger effects in EpAT (Figure 2b). Interestingly, although in body weight (Figure 2a). However, there were no differences in the weight of adipose depots did not differ, the number of

© 2015 Macmillan Publishers Limited International Journal of Obesity (2015) 782 – 790 SMI-mediated CD40-TRAF6 inhibition in obesity SM van den Berg et al 786 adipocytes per field in EpAT of mice treated with SMI 6860766 was SMI 6860766 does not induce differences in hepatosteatosis increased by 15.3%, revealing that SMI treatment decreased As liver steatosis is an important metabolic complication, we adipocyte size, suggesting less lipid storage and improved assessed liver weights, hepatic inflammation and degree of metabolic function (Figures 2c and d).23 A slight increase in the steatosis. However, in this relatively mild model of DIO (45% number of adipocytes per field in ScAT did not reach statistical calories from fat), we did not observe differences in liver weight significance (Figure 2c). between the groups (Figure 3a) or severe inflammation in either group (Figures 3b and c). Furthermore, there were no differences – SMI 6860766 improved glucose tolerance in DIO in hepatic lipid content and the degree of steatosis (Figures 3d f). Next, we evaluated the effect of SMI 6860766 treatment on glucose sensitivity. Basal levels of glucose and plasma insulin did Amelioration of AT inflammation after compound treatment not differ between the experimental groups and the HOMA-IR As CD40 is important in inflammation and AT inflammation has a indices were not different (Table 1). Glucose tolerance tests were key role in the development of insulin resistance, we analysed performed before and after 3 and 6 weeks of treatment. No immune cell composition by flow cytometry. We observed a 68.5% + differences between the groups were observed before treatment decrease in number of total leucocytes (CD45 cells) in the EpAT was initiated (data not shown). However, 3 weeks of treatment of mice treated with SMI 6860766 (Figure 4a). Leucocyte subset analysis revealed that T-helper cell numbers (CD3+CD4+ cells) with SMI 6860766 resulted in improved glucose sensitivity, + compared with vehicle-treated mice and an even more pro- were decreased by 50.4% and cytotoxic T-cell numbers (CD3 CD8+ cells) were decreased by 61.6%, whereas the numbers of nounced effect on glucose sensitivity was seen after 6 weeks of + + + + treatment (Figure 2e). These data indicate that SMI 6860766 regulatory T cells (CD3 CD4 CD25 FoxP3 cells) were not affected – improves glucose sensitivity in DIO. (Figures 4b d). No differences in ScAT T-cell numbers were observed (Supplementary Figures 2a–d). Additionally, the number of EpAT macrophages (CD11b+ Ly6G−F4/80+ cells) was decreased by 81.7% by SMI 6860766 Table 1. Characteristics of mice that received an HFD for 18 weeks (Figure 4e). Macrophage subset analysis showed that proinflam- + − + + − and were treated with vehicle or SMI 6860766 after 12 weeks of HFD matory M1 macrophages (CD11b Ly6G F4/80 CD11c CD206 and continued treatment until week 18 cells) were decreased by 80.8% and anti-inflammatory M2 macrophages (CD11b+Ly6G−F4/80+CD11c−CD206+ cells) by CTRL 6860766 70.5% (Figures 4f and g). The proinflammatory M1 macrophages ± ± showed a larger decrease than the M2 macrophages; therefore, Food intake (g per week) 20.9 0.6 22.5 1.1 the ratio between M1 and M2 macrophages was 2.4 in the control EpAT (mg) 1752 ± 203 1413 ± 206 ± ± group, whereas the treatment resulted in a ratio of 1.5, suggesting ScAT (mg) 1082 181 870 147 fl fi BAT (mg) 142 ± 10 136 ± 14 a more anti-in ammatory pro le of the EpAT depot (Figure 4h). No Glucose (mg dl − 1) 153 ± 6.6 157 ± 5.4 aberrations in ScAT macrophage numbers were observed Insulin (m U l − 1) 11.0 ± 0.99 11.1 ± 1.0 (Supplementary Figures 2e–g). HOMA-IR 4.2 ± 0.43 4.2 ± 0.39 Furthermore, immunohistochemical analysis of the immune cells in EpAT showed decreased amounts of leucocytes (CD45+)in Abbreviations: BAT, brown adipose tissue; CTRL, control; EpAT, epididymal AT; HFD, high-fat diet; HOMA-IR, homeostasis model assessment of insulin the SMI 6860766-treated group, which was not seen in ScAT resistance; ScAT, subcutaneous AT. N = 12; *Po0.05. (Figures 5a and b). Additionally, we observed a reduction in macrophage number (CD68+) relative to the number of adipocytes

Liver weight Leukocytes

Lipid content Macro steatosis Micro steatosis

Figure 3. No liver abnormalities were seen in vehicle or SMI 6860766-treated groups. (a) Liver weights did not differ. (b) Immunohistochemical leucocyte (CD45) staining and (c) scoring of hepatic inﬂammation by haematoxylin and eosin staining did not show signiﬁcant differences between the two experimental groups. (d) Oil red O staining of liver sections and (e and f) scoring of hepatosteatosis did not show differences in the degree of steatosis or lipid content (n = 12 per group).

Cytotoxic T cells Regulatory T cells

Macrophages M1 macrophages

M2 macrophages Ratio M1:M2 macrophages

Figure 4. Flow cytometric analysis demonstrates that SMI 6860766 treatment reduces AT leucocyte count. (a) Flow cytometry showed decreased leucocyte numbers (CD45+ cells) in the EpAT of SMI 6860766-treated mice. (b) Lymphocyte subset analysis revealed that, especially, T-helper (CD3+CD4+) cells and (c) cytotoxic (CD3+CD4−CD8+) T cells were decreased, whereas (d) regulatory (CD4+CD25+FoxP3+) T cells were not affected. (e) The number of total macrophages (CD11b+F4/80+) as well as (f) proinflammatory M1 (CD11b+F4/80+CD11c+CD206−) and (g) anti-inflammatory M2 (CD11b+F4/80+CD11c−CD206+) macrophages was decreased by treatment with SMI 6860766. (h) The ratio between M1 and M2 macrophages (n = 12 per group; *Po0.05 and **Po0.01). in EpAT of the SMI 6860766-treated group (Figures 5c and d). metabolic dysfunction.5,24,25 Interactions between adipocytes, Taken together, our data demonstrate that treatment with SMI T cells and macrophages result in the secretion of proinflamma- 6860766 ameliorated diet-induced AT inflammation. tory mediators, such as tumor necrosis factor and IL-6, which directly interfere with insulin signalling and promote insulin resistance.5,24,25 Strategies that deplete AT immune cells restore DISCUSSION insulin sensitivity.5,24,25 Skewing of immune cells towards their Immune cell activation and inflammation within the visceral AT proinflammatory subsets, such as M1 macrophages, is known to have a pivotal role in the pathogenesis of obesity and obesity- aggravate AT inflammation and insulin resistance.25,26 These associated insulin resistance.1,3,4 During the development of events change the AT into an insulin-resistant, proinflammatory obesity, the number of inflammatory cells, especially T cells and environment that propagates ongoing local and systemic meta- macrophages, increases and correlates with the extent of bolic deterioration.

EpAT ScAT

EpAT CD45

CCTRLTRL 68607666860766

EpAT

EpAT CD68

CCTRLTRL 68607666860766 Figure 5. Immunohistochemical assessment of AT leucocytes demonstrating reduced inﬁltration of total leucocytes and macrophages into AT of SMI 6860766-treated mice. (a) Immunohistochemical analysis of EpAT and ScAT by leucocyte (CD45) staining showing a decreased amount of leucocytes in the EpAT of SMI 6860766 treated mice. No differences were seen in leucocyte counts in ScAT of SMI 6860766-treated mice. (b) CD45 staining of EpAT illustrating the decreased amount of CD45+ cells in SMI 6860766-treated mice compared with vehicle. (c) Immunohistochemical analysis by macrophage (CD68) staining showing a decreased amount of macrophages in the EpAT of SMI 6860766- treated mice. (d) Pictures of a CD68 staining of EPAT to illustrate the decrease in CD68+ cells in SMI 6860766-treated mice (n = 12 per group; **Po0.01).

Costimulatory molecules are known to efficiently propagate and inflammation were significantly reduced upon antibody mediate immune reactions, and emerging evidence obtained treatment.30 Similar results were obtained when CD40L was from experiments in gene-deficient and transgenic mouse models blocked using an anti-CD40L antibody in DIO mice. Weight gain suggests that costimulatory molecules are an interesting was only slightly reduced, but glucose tolerance and insulin therapeutic target to combat obesity and the metabolic sensitivity did improve upon treatment, as did AT inflammation: syndrome.15,17–20,27–30 The therapeutic propensity of costimula- less CD4+ T cells and macrophages were present in the visceral tory molecules has been explored by treating DIO mice with AT.15 As genetic deficiency of CD40, the receptor for CD40L, antibodies against the CD28-CD80/86 and CD40L-CD40 aggravates obesity,18–20,28 Wolf et al.20 administered agonistic system.15,20,30 CD40 antibodies to DIO mice, and found that HFD-induced weight Simultaneous blocking of CD80 and CD86 by antibodies in DIO gain was abolished, glucose tolerance and insulin sensitivity had mice resulted in an improved glucose tolerance and in an increase improved and inflammatory cell recruitment (CD4+ T cells and M1 in insulin sensitivity. Moreover, AT inflammation and hepatic macrophages) in the visceral AT was halted.20 These data show

International Journal of Obesity (2015) 782 – 790 © 2015 Macmillan Publishers Limited SMI-mediated CD40-TRAF6 inhibition in obesity SM van den Berg et al 789 that antibody-mediated targeting of costimulation in obesity is a mediated inhibition compared with the effects that we previously powerful approach to improve inflammation-associated metabolic reported in the transgenic mice. Another explanation could be deterioration. However, although the results of antibody treat- that our SMI has other, not yet identified, beneficial effects on the ment are positive, long-term use of antibodies blocking costimu- inhibition of inflammation. latory receptors or ligands is likely to result in unwanted side In conclusion, we have shown that SMI 6860766 improves effects as long-term dysfunction of the CD40L-CD40 pathway or obesity-associated glucose sensitivity by reducing AT inflamma- CD80/86 pathway will result in immune system derangements. tion. SMIs that block the proinflammatory CD40-TRAF6 interaction In the present study, we therefore chose to block the CD40- are a promising therapeutic strategy. Additional development and CD40L system with a novel SMI that was generated to target the refinement of the inhibitors, as well as optimized delivery interaction between CD40 and TRAF6, and, we were able to methods such as nanoparticle-mediated drug delivery, are fl successfully improve AT in ammation and metabolic dysfunction. required before these SMIs can be used in humans. The small-molecule inhibitor approach has several advantages. By blocking only parts of the CD40 signal-transduction pathway, and not CD40 or CD40L itself, severe immune suppression will be CONFLICT OF INTEREST prevented. Moreover, our SMIs are selected using the FAF-Drugs2 The authors declare no conflict of interest. filter and the ADME/tox filter (Molécules Thérapeutiques In Silico, Paris, France) to obtain the most optimal administration, delivery, metabolism, extraction and toxicology profile,31,32 and in addition, ACKNOWLEDGEMENTS compounds containing reactive or toxic groups were rejected. We acknowledge the support from the Netherlands CardioVascular Research This screening process selected lead SMIs, which successfully Initiative: ‘the Dutch Heart Foundation, Dutch Federation of University Medical blocked inflammation and are very suitable for further drug Centres, the Netherlands Organisation for Health Research and Development and the development.19 Although the modelling and structure-based Royal Netherlands Academy of Sciences’ for the GENIUS project ‘Generating the best virtual ligand screening and validation was performed with great evidence-based pharmaceutical targets for atherosclerosis’ (CVON2011-19). This work care, it cannot be ruled out that our SMI is able to bind to other was supported by the Netherlands Organization for Scientific Research (NWO)(VICI proteins. grant to EL, and CW, medium investment grant to GN), the Netherlands Heart In a previous paper, we reported that we could improve insulin Foundation (Established investigator grant to EL, MW, Dr E Dekker grant to TS), the sensitivity, AT inflammation and hepatosteatosis by administering Rembrandt foundation (SB, MW, EL), the Deutsche Forschungsgemeinschaft (DFG FOR809: LU1643/1-2, SO876/3-1, WE1913/11-2, SFB914-B08 and SFB 1054 and our lead compound 6877002 (IC50 = 15.9 μM) during the past 6 weeks of the experiment in a model of DIO in which mice SFB-1123 to EL, CW, CH279/5-1 to TC), European Research Council Grants (No. 281296 19 to TC and ERC AdG No. 249929 to CW), a grant from the Else-Kroner-Fresenius- received a 60% HFD for 12 weeks. Here we used an optimized Stiftung (to TC) and by a grant from the German Federal Ministry of Education and SMI, derived from our lead compound, with an IC50 of 0.3 μM (SMI 19 Research to the German Center for Diabetes Research (DZD eV) (to TC) and DZHK 6860766), and used a milder, but a more prolonged model of (German Centre for Cardiovascular Research, MHA VD1.2 to CW), the Cardiovascular DIO (45% HFD for 18 weeks), which is more representative for the Research Institute Maastricht (to GN), the EU (Grant No. KBBE-2011-5 289350 to GN), clinical problem of obesity-induced metabolic complications. the Transnational University Limburg (to GN) and Cyttron II (FES0908 to GN). Treatment was started after 12 weeks of diet, when obesity was already evident, and was continued for 6 weeks. Remarkably, we saw a massive decrease in AT inflammation, with less influx of CD4 REFERENCES + and CD8+ T cells, as well as macrophages. This decrease was 1 Kanneganti T-D, Dixit VD. 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