IL-20 contributes to low grade inflammation and weight gain in the Psammomys obesus

International Immunopharmacology 45 (2017) 53–67

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IL-20 contributes to low grade inflammation and weight gain in the Psammomys obesus Helena Cucak 1, Lise Høj Thomsen 1, Alexander Rosendahl ⁎ Hagedorn Research Institute, DK-2820 Gentofte, Denmark

a r t i c l e

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Article history: Received 29 September 2016 Received in revised form 26 January 2017 Accepted 27 January 2017 Available online xxxx Keywords: Inflammation Lymphocytes Diabetes Cytokines Pre-clinical Obesity

a b s t r a c t The metabolic syndrome has been demonstrated in gene deficient animals, e.g. db/db mice, to include a systemic inflammation leading to insulin resistance, obesity and type 2 diabetes (T2D). To determine the importance of inflammation in obesity and diabetes, in a normal non-genetically modified species, an intervention study with neutralizing anti-IL-20 antibodies was conducted in the spontaneous T2D model Psammomys obesus. All IL-20 receptor chains were expressed on protein level in the Psammomys obesus. Neutralization of IL-20 did not modulate blood glucose, HbA1c, insulin levels or lymphocyte numbers after five weeks treatment although a trend to reduced weight gain rate was observed upon anti-IL-20 treatment. Inhibition of IL-20 significantly increased the number of CD11bhigh/low cells and the CD11bGr-1int myeloid derived suppressor cells in the spleen. Importantly, although the number of M1-like monocytes remained unchanged the M1-like marker CD11c expression level was reduced on the cells upon anti-IL-20 treatment. Anti-IL-20 treatment reduced both TLR4 and CCR2b expression on the macrophages upon treatment. Further, a marked shift in the protein signature in the pancreatic tissue after anti-IL-20 treatment was observed including enhanced expression of CXCL12, TIMP1 and IL-10 while IL-1β, CXCL4, PEDF and ADAMTS1 were reduced. In conclusion, we describe for the first time the systemic immune response in the diabetic Psammomys obesus. Neutralizing IL-20 modulated the myeloid compartment, the adaptive immunity, and local expression of proteins in the diabetic pancreatic tissue as well as improved on weight gain and hence may place IL-20 as a cytokine to be considered in obesity. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Type 2 diabetes (T2D) is a disease associated with obesity as a result of over-nutrition and low physical activity [1]. It is considered to be a heterogenetic disease associated with higher incidence with higher age [2]. Several genetic and epigenetic predispositions have been described to contribute to the disease development [3]. Classical hallmarks of the disease are a defect in insulin secretion and in established disease insulin resistance in peripheral organs like the liver, adipose tissues and skeletal muscle [4]. Recently, low grade inflammation characterized by elevated production of several cytokines and chemokines as well as local accumulation of inflammatory cells e.g. macrophages were shown to be present in T2D subjects and in pre-clinical models [5,6]. The local accumulation and activation of the immune cells has convincingly been shown to be a result of activation of pathogen receptors e.g. TLR4 by exogenous and endogenous factors which initiate local inflammation [7]. In adipose tissue the accumulated macrophages enhance the ⁎ Corresponding author at: Shire Inc., Tobaksvej 2, 2820 Soborg, Denmark E-mail address: [email protected] (A. Rosendahl). 1 Contributed equally to the present study.

http://dx.doi.org/10.1016/j.intimp.2017.01.031 1567-5769/© 2017 Elsevier B.V. All rights reserved.

inflammatory responses and contribute to the apoptosis of adipocytes which leads to peripheral insulin resistance [8]. During metabolic diseases circulating monocytes are directed towards various tissues due to distinct chemokine pathways [9]. Therein the monocytes specifically maturate and polarize into various forms of effector cells due to presence of various cytokines and growth factors in the tissue and migrate using specific chemokine receptors e.g. CCR2b [9]. The monocytes and macrophages express a combination of lineage markers like F4/80, CD68, CD11b and Ly6C, each partly specific for each organ and often associated with the effector function during the disease progression [5,6,10]. Further, a number of additional polarization markers among several CD11c and CD206 are often used to define M1-like and M2-like cells during metabolic diseases [11,12]. The diabetic islets of Langerhans show an accumulation of predominately phenotypically activated MHC class II and galectin-3 positive M1-like CD11c+ CCR2b+ macrophages [12]. This macrophage subset has an intrinsic enhanced capacity to produce several cytokines including IL-1β [13]. A positive feedback loop is present in the diabetic islets as the elevated glucose levels present in diabetes acts directly on the β-cells to release IL-1β [14]. β-cells respond to several pro-inflammatory cytokines in vitro by a reduction of insulin production per cell and apoptosis

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induction [15–17]. In pre-clinical models of diabetes inhibition of the IL1β pathway has shown some beneficial effects such as recovered β-cell function and improved glucose control [18,19]. The IL-10 cytokine family member, IL-20, is produced by activated keratinocytes and monocytes [20]. The functional receptor complex is a heterodimer complex of the alpha-chain IL-20RA and the beta-chain IL-20RB or the alpha-chain IL-22RA and the beta-chain IL-20RB expressed predominately on epithelial cells [21]. The biological functional of IL-20 includes regulation of proliferation, differentiation and local pro-inflammation [22]. Over-activity of IL-20 has been demonstrated in inflammatory conditions of the skin like psoriasis and rheumatoid arthritis [22]. In these diseases, IL-1β and TNFα have also been implemented to play as role [12,23]. Diabetic islets have significantly elevated expression of IL-20 and the IL-20 receptor chains and express high levels of several chemokines e.g. RANTES [24]. Recently, we described that IL-20 contributes to the systemic inflammation in the genetic leptin receptor deficient T2D mouse model db/db, where neutralization of IL-20 partly modulates the insulin resistance, weight gain and markedly modulates the systemic immune profile [24]. Moreover, the local inflammatory milieu in the diabetic pancreas was markedly modulated upon neutralization with anti-IL20 with reduced levels of RANTES and IL-16, but increased levels of MCP-1 and IL-6 [24]. Several pre-clinical models of obesity like the leptin deficient ob/ob mice and high fat diet mice/rats and T2D models like the leptin receptor deficient db/db mice and zucker-rats have been used to evaluate development of disease and to demonstrate effect of novel therapeutics [25, 26]. However, while these models capture several aspects of the disease caution should be taken when evaluating the immune response in the leptin deficient models as these animals are highly immune compromised which is not the case in the human patient population. Moreover, while the HFD models develop peripheral insulin resistance, they do not develop beta cell death and as such are poor models for T2D [27]. The Psammomys obesus is a terrestrial mammal from the gerbil subfamily normally lives in the desert in North Africa and the Middle East which just recently has been recognized as a new pre-clinical laboratory species and hence is not yet fully explored on the genome level [28]. This non-genetically manipulated animal rapidly acquires a metabolic disease closely resembling T2D when feed ordinary rodent chow in laboratory environment. This phenomenon of short disease progression in a genetically normal animal has recently drawn attention to this model as a good model to evaluate T2D and future treatment effects [28].

Given the potential opportunity to evaluate inflammatory diseases like diabetes and obesity in a non-genetically modified animal, we performed a systematic but limited evaluation of cross-reactivity of wellknown FACS reagents in the Psammomys obesus spleen. As the reagents used are developed against mouse and human antigens, our data provides in addition to novel biological understanding also novel tools to evaluate inflammatory pathways in a non-genetically modified obesity/diabetes pre-clinical species. The growing evidence suggesting that inflammation contributes to T2D progression led us to evaluate the importance of the IL-20 axis in the pre-clinical Psammomys obesus model of T2D. Our data demonstrate that the neutralizing anti-IL-20 does not provide a significant effect on metabolic parameters like HbA1c, blood glucose in the Psammomys obesus. However, inhibition of IL-20 potently modulates the low grade inflammation mediated by both the innate and adaptive immunity that may partly regulate weight gain and obesity. 2. Material & methods 2.1. Ethics statement All animal experiments were approved by the Copenhagen Animal Ethics Committee and performed according to their recommendations. 2.2. In vivo evaluation of anti-IL-20 effect in Psammomys obesus Male Psammomys obesus were obtained from Harlan Laboratories Ltd. Jerusalem at the age of 7–10 weeks and acclimatized for 4– 5 week before start of the experiment. Only male animals were used in the experiments as they more robustly than the female animals developed diabetes. On arrival the animals were feed low energy diet (LE-diet) (3084–111,507, 2.5 kcal/g, Harlan Teklad). Base line blood glucose was measured after acclimatization period ended and hereafter the animals were fed high energy diet (HE-diet) (Formulab Diet 5008, 3.5 kcal/g, Lab Diet) for an additional 10 days. All feeding was ad libitum. Diabetes development was evaluated by measuring blood glucose after 0, 2, 4, 7 and 9 days on HE diet. Then the animals are put back on LE-diet until the initiation of the experiment. 35 responders (developed diabetes) were selected and included in the experiments and randomly assigned to 3 equally sized groups of animals. All animals are changed to high energy diet HE-diet at the same time as the treatment was initiated. The IgG4 1400–250-5B7 anti-human IL-20 antibody previously

Table 1 Antibodies used in flow cytometric analysis. Antigen

Flourochrome

Vendor

Cat. Number/Clone name

Functionality in the Psammomys obesus

B220 NK1.1 CD4 CD11b Gr-1 CD11c TLR2 TLR4 F4/80 CD3 CD8a IL-21R CXCR3 CCL2 IL-20R beta Anti-sheep IgG IL-22RA

V500 APC/Cy7 Pacific Blue PE-Cy7 PE APC Alexa F647 PE APC-Cy7 Alexa F488 PerCP PE PE PE Purified PE Purified PE PE Alexa F647 Alexa F488

BD Biosciences Biolegend Biolegend Biolegend R&D Systems BD Biosciences eBiosciences BD Biosciences Biolegend BD Biosciences BD Biosciences R&D Systems eBiosciences Biolegend R&D Systems abcam Millipore BD Biosciences R&D Systems BD Biosciences Biolegend

561226/RA3-6B2 108724/PK136 100531/RM4-5 101216/M1/70 FAB1037P/RB6-8C5 550261/HL3 51-9021/6C2 558294/MTS510 1231118/BM8 557666/145-2C11 553036/53-6.7 FAB5961/155516 12-1831/CXCR3-173 505904/2H5 AF4388/PolyclonalSheepIgG Ab7009 06-10777 PolyclonalRabbitIgG 558416 FAB11762P/173714 558406/48607 141710/C068C2

No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No

IL-20R alpha CCR2b CD206

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Fig. 1. Expression of IL-20 receptors on splenocytes in Psammomys obesus. Splenocytes were stained with antibodies towards IL-20RA, IL-20RB and IL-22RA and the corresponding isotype controls at 3-fold higher concentration. The relative mean fluorescence on the splenocytes (A) and the corresponding frequency of positive cells above isotype control (B) was determined for each IL-20 receptor subunit. Gating strategy to identify the receptor positive cells is shown (C) SSC/FSC, (D) doublet exclusion (E), dead-cell exclusion, (F) IL-20RA antibody, (G) IL-20RB antibody, (H) IL-22RA antibody, (I) IL-20RA isotype, (J) IL-20RB isotype and (K) IL-22RA isotype. Each experiment was performed two times and in each group three individual animals were stained. Statistical evaluation was performed with 1-way ANOVA.

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that show cross-reactivity to mouse IL-20 [29] or vehicle were injected once weekly, day 1, 8, 15 and 22 at a concentration of 20 mg/kg i.p with a volume of 5 ml/kg. At day 22 the animals were terminated by cervical dislocation in isoflurane.

measured in a Biosen S_Line, auto-analyzer (EKF Diagnostics GmbH, Germany) according to the manufacturer's instructions.

2.3. Blood glucose measurement

HbA1c was measured once weekly in 5 μl full blood sample taken from the tip of the tail by puncturing the capillary bed with a lancet, using a heparinized capillary tube to sample the blood. The blood was shaken into 500 μl Hitachi Hemolyzing Reagent and measured in a Hitachi 912 auto-analyzer (Roche A/S Diagnostics, Germany) according to the manufacturer's instructions.

Blood glucose was measured twice/week in 10 μl full blood sample taken from the tip of the tail by puncturing the capillary bed with a lancet, using a 10 μl heparinized capillary tube to sample the blood. The blood was then shaken into glucose/lactate System Solution and

2.4. HbA1c measurement

2.5. Plasma insulin measurement Insulin levels were determined in blood by the luminescence oxygen channeling immunoassay (LOCI) [15]. Briefly, blood taken in capillary tubes was centrifuged in hematocrit centrifuge. 10 μl plasma was transferred directly to micronic tubes. Detection of insulin was by luminescence oxygen channeling immunoassay (LOCI). Anti-insulin mAb RDITRK2IP10-D6C4 was conjugated to LOCI acceptor beads (PerkinElmer) and another anti-insulin mAb RDI-TR- K2IP10-D3E7 (binding to a different epitope) was biotinylated. The assay was conducted in 384 well plates by adding 1 μl of calibrator, control and unknown sample in the wells followed by 15 μl of a mixture of acceptor beads and biotinylated antibody. After 1 h of incubation at 21–22 °C, 30 μl of streptavidin-coated donor beads were added and the plates were further incubated for 30 min. The plates were read in an Envision plate reader (PerkinElmer) at 21–22 °C, applying a 520–645 nm filter after excitation by a 680 nm laser. 2.6. Expression of inflammatory proteins in the pancreatic tissue Snap frozen pancreas was homogenized in presence of protease inhibitors (10 μg/ml Aprotinin, 10 μg/ml Leupeptin, and 10 μg/ml Pepstatin) and 1% Triton X-100 to obtain pancreatic protein lysates. By centrifugation (10,000 rpm for 10 min) cellular debris was removed. Protein concentration was determined by colorimetric detection by use of the BCA Protein Assay Kit (Thermo Fischer) where equimolar amount from each group was added to proteome angiogenesis array profiler membranes (ARY015) and protein expression was evaluated according to manufacturer's description (R&D Systems). Relative expression was determined using Image J software and fold induction shown compared to vehicle treated Psammomys obesus. 2.7. Serum cytokine analysis Serum levels of TNFα (R&D MTA00B), IL-6 (R&D M6000B), IFNγ (MIF00), IL-1β (R&D MLB00C) and IL-10 (R&D M1000B) were determined using manufacturers descriptions. 2.8. Flow cytometry analysis of splenocytes in the Psammomys obesus

Fig. 2. Metabolic effect and oral glucose test after anti-IL-20 treatment. One week post last treatment with anti-IL-20, the animals was terminated. During the experiment and at time of termination blood glucose (A), HbA1c (B) and plasma insulin levels (C) were determined. Each group contained 12 animals. Statistical evaluation was performed with 2-way ANOVA.

Eight color flow cytometric analysis was performed according to standard procedures. Specificity of the antibodies used was determined with appropriate isotype controls and relevance of the staining's verified by population having the expected FSC-SSC pattern. Briefly, spleens were removed and single cell suspension was generated. Cells were blocked using anti-mouse CD16/CD32 antibodies (BD PharMingen, San Diego, CA, USA), followed by staining with primary and if required secondary antibody according to Table 1. In case of intracellular staining's, cells were fixed and permeabilized using the Cytofix/ Cytoperm fixation/permeabilization solution kit (BD PharMingen), according to the manufacturer's description and then intracellularly stained with antibodies as above. Samples were then acquired on a FACS LSRFortessa, equipped with blue, red, and violet laser, followed by data analysis using FACSDiva software (BD Biosciences, San Jose,

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2.10. RNA extraction and qPCR analysis RNA was extracted from splenocytes with RNeasy Lipid Tissue Mini Kit as described by the manufacturer's instructions (cat#74804, Qiagen, Denmark) and concentration determined by Nanodrop and cDNA was made using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems). Gene expression was analyzed on TaqMan Array Card (Life Technologies) using TaqMan Gene Expression Master Mix (Applied Biosystems). The primer ID used were CD206 (Mm00485148_m1), Arg1 (Mm00475988_m1) and Mgl (Mm00546124_m1).

3. Results 3.1. IL-20 receptor is expressed in diabetic Psammomys obesus spleens The sequences of the IL-20 receptors are not described in the Psammomys obesus. In order to determine the expression of the IL-20 receptors, a flow cytometric analysis of splenocytes was conducted. Currently, no reagents are developed that specifically recognize Psammomys obesus antigens. For this reason, we evaluated expression using mouse reactive antibodies (Table 1) and corresponding isotypes on living cells by excluding 7AAD positive cells. The reactivity of all antibodies used is shown in Table 1. A low immunoreactivity was demonstrated in 0.3% of the splenocytes when stained with anti-human IL-20RA antibodies (Fig. 1B, F). At 3-fold excess of an isotype antibody, the IL-20RA antibody signal was still significantly higher suggesting that the signal obtained was specific rather than unspecific stickiness (Fig. 1A, F, I). Both the IL-20RB and the IL-22RA antibodies showed a higher mean fluorescence signal and higher signal to noise compared to the isotype control antibodies (Fig. 1A, G, H, J, K). Close to 1% of the splenocytes were IL-20RB positive, while not N0.2% showed specific IL-22RA expression (Fig. 1B). Although the frequency of positive cells was low, the significantly elevated expression compared to 3-fold access of corresponding isotype and given that the reagents has been shown to recognize the receptor chains specifically in transfected cell lines verify that the signals and receptor positive populations represents real populations. These data demonstrate that all the IL-20 receptor complexes are present in the Psammomys obesus. Based on the morphological expression pattern of the IL-20RA and IL-20RB the cells may belong to the same subset and hence co-express functional receptors providing support for further in vivo evaluation of the functional importance of the cytokine in disease. Fig. 3. Body weight effects after neutralizing anti-IL-20 treatment. Animals were weighted twice per week until termination one week post the last treatment with anti-IL-20 antibodies. The actual weight of each animal was plotted against time in the experiment (A) as were the weight gain in grams per animal during the experiment (B) and weight increase in percentage to each individual animals starting weight (C). Each group had 12 mice. Statistical evaluation is performed with 1-way ANOVA (A, B) or t-Test (C).

CA, USA). The absolute number of cells was determined through 123count eBeads™ from eBioscience using manufactures instructions. The number of viable cells obtained from single-cell pancreatic tissue is very limited and no standard protocol is developed to be used in the Psammomys obesus. Therefore flow cytometric analysis after neutralization with anti-IL20 could not be performed locally in the pancreatic tissue and were therefore conducted in the systemic spleen compartment.

2.9. Flow cytometry analysis of islets in the Psammomys obesus Three color flow cytometric analysis was performed according to standard procedures [12]. Briefly, pancreatic tissue from 5 animals was removed and single cell suspension was generated using the cold collagenase method described in [15].

3.2. Neutralizing anti-IL-20 does not modulate development of diabetes Based on the presence of the IL-20 receptors in the Psammomys obesus, the effect on diabetes development and insulin production was evaluated after treatment with neutralizing antibodies towards IL-20. Within five days after initiation of treatment, the blood glucose levels increased significantly (p b 0.0001) 3-fold in the vehicle treated group (Fig. 2A). After 10 days the maximum blood glucose level (4fold basal level) was reached (Fig. 2A). Treatment with anti-IL-20 did neither influence the early raise in blood glucose nor the maximum blood glucose level obtained (Fig. 2A). A continuous significant increase of HbA1c in the Psammomys obesus was demonstrated already after 10 days which continued to increase with 0.5% every 10 days during the experimental period (Fig. 2B). Neutralizing anti-IL-20 did not influence the initial raise or the slope of the HbA1c (Fig. 2B). At the end of the experiment, the plasma levels of insulin were similar in the neutralizing anti-IL-20 treated and the vehicle group (Fig. 2C). These data demonstrate that neutralizing anti-IL-20 treatment for 5 weeks in the Psammomys obesus had no effect on development of diabetes measured as blood glucose, HbAc1 or insulin production.

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Fig. 4. Flow cytometric evaluation of monocyte and macrophage populations in the Psammomys obesus spleen after treatment with anti-IL-20. At termination of the experiment, the spleen was removed and total number of splenocytes (A), frequency (B, D, F) and actual numbers (C, E, G) was determined for CD11bhigh (B, C), CD11blow (D, E) and F4/80 (F, G) cells. Each experiment was performed two times and three representative animals based on metabolic parameters were analyzed in each group. Statistical evaluation is performed with 1-way ANOVA.

3.3. Neutralizing anti-IL-20 partly modulate weight gain in Psammomys obesus As cytokines have been associated with both weight gain and weight loss, the effect on the weight was evaluated in Psammomys obesus after neutralizing of IL-20 [30].

Vehicle treated animals showed a rapid onset of weight gain that was continued throughout the treatment period with a weight gain of 1.025 g per day (0.7758–1.274 g; 95% confidence interval) (Fig. 3A). Anti-IL-20 treated Psammomys obesus gained weight throughout the treatment with a weight gain of 0.8415 g per day (0.6225–1.061 g; 95% confidence interval). To correct for the weight on an individual

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Fig. 5. Frequency of CCR2b positive monocytes and macrophages sub-populations in the Psammomys obesus spleen after treatment with anti-IL-20. At termination of the experiment, the spleen was removed and the frequency of CCR2b positive CD11bhigh (A), CD11blow (B) and F4/80+ (C) cells was determined. Each experiment was performed two times and three representative animals based on metabolic parameters were analyzed in each group. Statistical evaluation is performed with 1-way ANOVA.

level, the basal starting weight (2 days prior to the start of the experiment) was subtracted from each individual. On average the vehicle group gained 33.4 g during the 35 day treatment period with an individually corrected slope of 1.095 g/day (0.9041–1.106 g; 95% confidence interval) (Fig. 3B). The anti-IL-20 treated animals in contrast gained 27.5 g on average during the treatment period with an individually corrected slope of 0.8311 g/day (0.7108–0.9514 g; 95% confidence interval) (Fig. 3B). The corrected weight gain did not reach significance (p = 0.0796) at the end of the experiment, but a clear trend to a reduce weight gain rate was noted during the treatment period (Fig. 3B). Most interestingly, when comparing the percentage weight increase per individual animal, neutralizing anti-IL20 did significantly reduce the weight gain (p = 0.0384) (Fig. 3C). These data demonstrate that although neutralizing anti-IL-20 treatment did not significantly modulate the weight during the 5 weeks treatment in the Psammomys obesus. However, a trend to weight gain reduction (in grams), reduced weight gain rate and reduced percentage weight gain was observed. 3.4. Neutralization of IL-20 modulates the monocyte populations in the spleen and in adipose tissue As IL-20 is produced by activated monocytes, which are present in the spleen of diabetic animals, and is known to modulate inflammation in other diseases, the effect on the monocyte and macrophages was determined in the spleen and adipose tissue by flow cytometric analysis. Monocytes and macrophages can be identified using many different markers [9]. Our study, using a small population of animals, focused on some of the most commonly used general phenotypes of monocytes/macrophages and the corresponding gating strategy of these monocyte populations are shown in supplementary Fig. 1 (Supplementary Fig. 1A–D). The overall cellularity in the spleen increased significantly in Psammomys obesus receiving anti-IL-20 neutralizing antibodies (Fig.

4A). This translated into a significant (p = 0.0009 and p = 0.0046 respectively) increase of the total number of CD11bhigh and CD11blow cells (2-fold and 1.5-fold respectively) after anti-IL-20 treatment (Fig. 4C, E). However, the frequency of CD11bhigh or CD11blow cells in the spleen was not modulated upon treatment (Fig. 4B, D). In contrast, while the total number of F4/80 macrophages remained unchanged upon treatment the frequency of F4/80 positive macrophages in the spleen was significantly (p = 0.0102) reduced after neutralizing antiIL-20 treatment compared to vehicle treated animals (Fig. 4F, G). Gating strategies for CCR2b expression uncovered significant differences in the expression in these macrophage subpopulations (Supplementary Fig. 1E–M). Approximately 10% of the CD11bhigh cells and 50% of the CD11blow cells were CCR2b positive and neutralization of IL-20 did not modulate the CCR2b expression (Fig. 5A, B). In contrast, close to 80% of the F4/80+ macrophages were CCR2b positive in the vehicle treated Psammomys obesus while neutralization of IL-20 significantly reduced the frequency of positive cells (Fig. 5C). A small evaluation of the CD45+ leukocytes in adipose tissue showed the majority of these cells to be F4/80+/CD11b+ monocytes expressing CCR2b in the vehicle treated Psammomys obesus (Supplementary Table 1A). No significant changes were demonstrated after neutralization with anti-IL20 antibodies (Supplementary Table 1A). Taken together, these data demonstrate that neutralizing IL-20 antibodies modulates the total number and the frequency of CCR2b positive monocytes in the diabetic spleen of Psammomys obesus. 3.5. Neutralization of IL-20 reduces M1-like polarization of macrophages and promotes development of myeloid suppressor cells As neutralization of IL-20 modulated the monocyte populations in the spleen, the macrophage polarization profile and presence of myeloid derived suppressor cells (MDSCs) was determined by flow cytometric analysis and the gating strategy for these populations is shown in supplementary (Supplementary Fig. 2).

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Fig. 6. Presence of M1-like and MDSC cells in the Psammomys obesus spleen after IL-20 treatment. At termination of the experiment, the spleen was removed and frequency (A, B) and actual numbers (C, D) was determined for M1-like CD11bhigh-CD11chigh (A, C), MDSC-like CD11bhigh-Gr1int (B, D) cells. Mean fluorescence intensity of CD11c on CD11b monocytes (E). Each experiment was performed two times and three representative animals based on metabolic parameters were analyzed in each group. Statistical evaluation is performed with 1way ANOVA.

Up to 13% of the CD11b cells in the vehicle treated Psammomys obesus spleen expressed high levels of CD11c; indicative of a M1-like phenotype (Fig. 6A). Treatment with neutralizing anti-IL-20 antibodies significantly (p = 0.0192) reduced the frequency of these M1-like macrophages (Fig. 6A). Despite the overall increase of the number of CD11b cells in the anti-IL-20 treated Psammomys obesus spleen, no increase of the total numbers of CD11c+ M1-like macrophages after treatment was observed (Fig. 6C). In fact, the overall surface expression level of CD11c was significantly reduced on the CD11b cells by anti-IL-20 treatment

Table 2 Fold induction of M2-like mRNA transcripts in spleen. Gene

Fold change Anti-IL20 vs vehicle

Arg Mgl CD206

319 ± 55 26 ± 14 N.D.

(Fig. 6E). CD206 was used as a marker for M2-like cells, but did not show cross-reactivity towards the Psammomys obesus preventing further evaluation of the M2-like phenotype. The frequency of moderately granulated CD11bGr-1int MDSC was not modulated by treatment with anti-IL-20 (Fig. 6B). In contrast, neutralization of IL-20 significantly (p = 0.0064) increased the total number of CD11bGr-1int MDSC in the Psammomys obesus spleen (Fig. 6D). The fact that these CD11bGr-1int cells were clearly distinct from the highly granulated CD11bGr-1high neutrophils strongly suggests that these CD11bGr-1int cells were MDSC rather than neutrophils (Supplementary Fig. 2C–D). A small mRNA evaluation of M2-like markers on whole spleen tissue showed no cross-reactivity of CD206 with the mouse reagent, while both Arg1 and Mgl cross-reacted to Psammomys obesus and were both up-regulated after anti-IL20 neutralization (Table 2). Taken together, these data suggests that neutralizing IL-20 antibodies counteracts the M1-polarization, upregulate M2-like genes and promotes development of MDSC in the spleen.

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Fig. 7. Flow cytometric evaluation of the expression profile of TLR2 and TLR4 receptors on monocytes in the Psammomys obesus spleen after IL-20 treatment. At termination of the experiment, the spleen was removed and mean fluorescence of TLR2 (A, D, G) and TLR4 (B, E, H) was determined on F4/80+ cells (A, B, C) CD11bhigh (D, E, F) and CD11blow (G, H, I). Representative FACS dot-plots of TLR4 expression on F4/80+ (C), CD11bhigh (F), CD11blow (I) were shown in vehicle animals (blue) and anti-IL-20 treated (red). Each experiment was performed two times and three representative animals based on metabolic parameters were analyzed in each group. Statistical evaluation is performed with 1-way ANOVA. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.6. Modulation of TLR expression after neutralization of IL-20 Apoptosis of Langerhans β-cells is dependent of excessive TLR signaling on macrophages during diabetes [15]. To determine if the IL-20 axis regulates the capacity of the innate immunity, expression levels of TLRs was determined by flow cytometric analysis. The expression level of TLR4 was high on vehicle treated F4/80 and CD11blow cells, whereas it was moderate on CD11bhigh cells (Fig. 7B, E, H). TLR2 expression was high on F4/80, but moderate on both the CD11blow and CD11bhigh cells in Psammomys obesus spleen (Fig. 7A, D, G). Both F4/80 macrophages and CD11blow cells responded to anti-IL20 treatment by a clear trend to down-regulation of TLR4, although not reaching significance (Fig. 7B, H). In contrast, CD11bhigh cells showed a significant down-regulation of TLR4 surface expression after anti-IL-20 treatment (Fig. 7E). No modulation of TLR2 expression level was observed on F4/80, CD11blow or CD11bhigh cells after neutralization of IL-20 (Fig. 7A, D, G). Taken together, these results demonstrate that inhibition of IL-20 modulates the expression level of TLR4, but not TLR2, suggesting that the responsiveness to exogenous endotoxins like LPS and endogenous TLR4 ligands like heat shock protein (hsp) might be modulated by IL20 in the diabetic Psammomys obesus.

3.7. Neutralization of IL-20 counteracts granulocyte but promote DC development To evaluate if neutralizing anti-IL-20 treatment modulates other cell types, the presence of neutrophils and DC-like cells was evaluated by flow cytometric analysis. A small population of CD11cveryhigh cells has previously been demonstrated to belong to the DC lineage whereas the Gr-1veryhigh cells have been shown to be granulocytes mainly neutrophils using the same reagents as in this study [31,32]. Gating strategy of the highly granulated Gr-1veryhigh neutrophils and the DC CD11cveryhigh cells is shown in Supplementary Fig. 3. The frequency of DC-like cells was not modulated, while a significant increase of the total number of the cells was observed after neutralization of IL-20 (Fig. 8A, B). In sharp contrast, a 2-fold significant reduction of the frequency of neutrophils was demonstrated after neutralizing anti-IL-20 treatment (Fig. 8C, Supplementary Fig. 3). This reduced frequency was translated into a significant reduction of the total number of neutrophils in the spleen after treatment (Fig. 8D). Taken together, neutralizing IL-20 antibody treatment reduced the neutrophil population while promoting DC development in the Psammomys obesus spleen.

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Fig. 8. Flow cytometric evaluation of neutrophils and dendritic cells in the Psammomys obesus spleen after IL-20 treatment. At termination of the experiment, the spleen was removed and frequency (A, C) and total number (B, D) of CD11cveryhigh DC-like cells (A, B) and Gr-1veryhigh neutrophils (C, D) were determined. Each experiment was performed two times and three representative animals based on metabolic parameters were analyzed in each group. Statistical evaluation is performed with 1-way ANOVA.

3.8. Anti-IL-20 modulates the lymphoid composition in the spleen With the observed modulation of the myeloid cell compartment after anti-IL-20 treatment, the effect on T cells, B cell and NK cells was evaluated by flow cytometry in the Psammomys obesus spleen. Antibodies towards B cells showed no cross-reactivity towards Psammomys obesus preventing evaluation of this cell compartment. The distribution of CD4 and CD8 T cells within the CD3 positive T cells in the vehicle treated Psammomys obesus showed comparable frequency as previously described in the diabetic db/db mouse, but up to 35% of the CD3+ cells did not express neither CD4 or CD8 in the Psammomys obesus (Fig. 9A, C) [12]. Treatment with neutralizing IL-20 antibodies significantly reduced the frequency of CD4+ T cells, but did not modulate the absolute numbers of CD4+ T cells in the spleen (Fig. 9A, B). Neither the frequency nor the absolute numbers of CD8+ T cells were modulated by anti-IL-20 treatment (Fig. 9C, D). The frequency of NK cells was significantly reduced by anti-IL-20 treatment, but this did not translate into a reduction of the absolute numbers of NK cells (Fig. 9E, F). Importantly, as this NK1.1.+ NK population is absent in the db/ db diabetic mouse, the Psammomys obesus is likely to be the first spontaneous pre-clinical type 2 diabetic model allowing evaluation of NK cell importance in type 2 diabetes. Taken together, these results show the diabetic Psammomys obesus to have a similar composition of T cells and NK cells as the diabetic db/db mouse. Neutralization of IL-20 modulates the cellular dynamics of CD4 and NK cells but not CD8 T cells in the spleen. 3.9. Neutralizing anti-IL20 counteract development of a systemic pro-inflammatory cytokine signature In order to determine if systemic cytokine signature is modulated after IL-20 neutralization, plasma from Psammomys obesus was

evaluated for presence of a number of cytokines known to be pro-inflammatory or resolving in nature. Overall, only low levels of cytokines were detected in the systemic circulations in the Psammomys obesus (Fig. 10). In fact, even after assay optimization individual animals barely reached detectable levels and IFNγ could not be detected. It is not possible to exclude that the low level observed is due to poor species crossover or reflects low levels present in the serum of the cytokines. Despite this, a clear trend to reduced levels of IL-1β and a concomitant increased level of IL-10 was noted in the anti-IL-20 neutralizing animals compared to the vehicle treated animals (Fig. 10). Taken together, our data show that the levels of circulating cytokines are low and seem to be modulated towards a repair/remodeling signature after anti-IL-20 neutralization. 3.10. The inflammatory and tissue remodeling protein signature in the pancreatic tissue is influenced by IL-20 In order evaluate the effect neutralizing anti-IL-20 treatment has on local inflammatory and tissue remodeling proteins in the diabetic pancreas a protein array study was performed on whole pancreas tissue from vehicle treated diabetic and anti-IL-20 treated Psammomys obesus animals. The low yield of cells per Langerhans islet prohibited local FACS analysis after treatment with anti-IL20, but the leukocyte composition in non-treated animals showed presence of predominately CD68+ leukocytes (Supplementary Table 2). Treatment with neutralizing anti-IL-20 significantly modulated the expression levels both by enhancing and reducing expression levels of specific proteins (Table 3). Five of the investigated proteins were significantly upregulated, while 22 proteins were significantly down-regulated upon anti-IL-20 treatment (Table 3). Interestingly, the chemokine SDF-1 (CXCL12) was the most upregulated proteins showing 2-fold

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Fig. 9. Flow cytometric evaluation of lymphocytes in the Psammomys obesus spleen after IL-20 treatment. At termination of the experiment, the spleen was removed and frequency (A, C, E) and total number (B, D, F) of CD4 T cells (A, B), CD8 T cells (C, D) and NK cells (E, F) was determined. Each experiment was performed two times and three representative animals based on metabolic parameters were analyzed in each group. Statistical evaluation is performed with 1-way ANOVA.

enhanced levels upon anti-IL-20 treatment (Table 3). Further, highly significant upregulation of TIMP-1, angiogenin and endothelin-1 was noted (Table 3). Of note, the cytokine IL-10, belonging to the same cytokine family as IL-20, also showed significant upregulation (Table 3). At the other end, IL-1β, platelet factor 4 (CXCL4), PEDF and ADAMTS1 were all highly significantly down-regulated (p b 0.0001) 5–10-fold compared to vehicle treated animals (Table 3).Other proteins showing significant down-regulation included among others the inflammatory factors like CX3CL1, MIP1α (CCL3), GM-CSF, CD105, the insulin-like growth factor binding proteins IGFBP1 and IGFBP3, tissue remodeling factors like TIMP4, pentraxin-3, EGF and HB-EGF (Table 3). Taken together, these data shows that neutralization of the IL-20 cytokine with systemic administration of antibodies markedly modulates local expression of pro-inflammatory and tissue remodeling proteins in the diabetic pancreatic tissue.

4. Discussion The new lifestyle with reduced physical activity and excess intake of calories and fat has led to a steady increase in the number of obese individuals with N65% of the adult population in the US currently being overweight and obese [33]. With the strong correlation between obesity and development of type 2 diabetes (T2D) is therefore of great concern as N330 million patients at present are diagnosed with T2D [34]. Although new agents to minimize the obesity such as the GLP-1 agonists show promising clinical results, the need to have add-on therapies to the increasing number of T2D patients is high [35]. Both obesity and T2D have an aberrant inflammatory activity, the low grade inflammation, which is still present even after the first-line therapy [6]. Our demonstration that neutralization of IL-20 modulates both the innate immunity and aspects of the adaptive immunity is

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Fig. 10. Systemic cytokine signature after IL-20 inhibition. At the end of the experiment the plasma levels of TNFα (A), IL-6 (B), IL-1β (C) and IL-10 (D) were determined. Each group had 12 animals and statistical evaluation was performed with 1-way ANOVA.

hence intriguing. Improved understanding of the underlying mechanisms of insulin resistance in adipose tissue has shown a key role for the inflammatory macrophage [36]. This cell type immigrates into the adipose tissue and initiates production of pro-inflammatory cytokines contributing to dysfunction of the tissues. In addition to the adipose tissue, these classically activated M1-like cells have been described in pancreatic tissue, skeletal muscle and in the diabetic kidney where they contribute to the progression of the disease [12,36–38]. There is strong evidence that inflammation contributes to development of diabetes in pre-clinical species [39]. However, most of these studies has been performed in genetically modified species and has shown poor translation into human trials on diabetic development although they show clear effects on macrovascular parameters also in human subjects. Based on these observations from various pre-clinical models and human studies it is intriguing that anti-IL-20 in fact significantly increases the number of splenocytes rather than leads to a reduction in cell number in the non-genetically modified Psammomys obesus. However, our in-depth analysis of the splenocyte macrophage pool that revealed that the frequency of the classically activated M1-like cells was reduced after neutralization of IL-20 suggests that the cellular balance between the classically and the alternatively activated macrophages might be shifted towards the latter upon treatment. The fact that the number of M1-like macrophages was not changed although the CD11b population was significantly enhanced suggests that the expansion rather indicate an expansion of the immune-modulating M2-like phenotype. This alternatively activated macrophage sub-population produces factors participating in the resolution of the inflammatory conditions and contributes to tissue repair [40]. In fact several studies has shown that the number of M2-like macrophages increase with exercise which reduce or even counteract development of obesity [41]. As the M2-like CD206 antibody did not cross-react to the Psammomys obesus confirmation of this hypothesis is not possible within the current study. Cancer patients often has a sub-population of bone marrow derived myeloid derived suppressor cells (MDSCs) which possess a broad array

of inhibitory and suppressive functions including inhibition of CD4-mediated immunity and cytokine production in macrophages [42–44]. In a normal mouse, a small population of 3–5% of the splenocytes co-expresses the αM-integrin CD11b and the Gr-1/Ly6G antigen which is used to identify the MDSC [45]. In the Psammomys obesus diabetic spleen, approximately 6% of the cells were CD11b-Gr1 cells. This frequency was not modulated by neutralizing of IL-20. Most importantly, the total numbers of the MDSC was however increased 2-fold by neutralizing of IL-20. Many cytokines have been shown important for regulation and growth of hematopoietic cells [46]. IL-20 synergize with colony stimulating factor (CSF) to increase the size of the colonies formed, but does not directly induce colony formation of CD34+ precursor cells [47]. IL-20 stimulation of the colonies promotes mainly erythrocytes and megakaryocytes rich colonies, although some precursors for granulocytes and monocytes were also observed [47]. As the number of MDSC was increased upon IL-20 neutralization, absence of IL-20 altered the release of immature cells from the bone marrow, promoted the peripheral proliferation or inhibited the apoptosis of these cells. MDSC cells co-cultured with CD4 + T cells in vitro or injected into mice potently inhibit proliferation of the CD4+ T cells [48]. The significantly reduced CD4+ T cell population observed in the Psammomys obesus after neutralization of IL-20 might hence be a result of MDSC mediated inhibition of CD4 proliferation. Of interest, 35% of the CD3+ cells in the Psammomys obesus were CD4−/CD8−. An elevated frequency of peripheral CD4−/CD8− cells are present also in the diabetic db/db mouse [12]. These systemic CD4−/CD8− T cells has in various models been associated with regulatory functions promoting tissue remodeling rather than extensive inflammation [49]. Whether the elevated CD4−/ CD8− cell population noted in the Psammomys obesus is related to regulatory functions or due to low cross reactivity of the reagents cannot be determined in this study. Obese patients have elevated levels of circulating endotoxins [50]. The endotoxins like LPS binds to the Toll-like receptors (TLR) and initiate robust pro-inflammatory cytokine production shown to promote

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Table 3 Heat map plot of the relative expression of pancreatic proteins in Psammomys obesus at the termination of the experiment. Proteins up-regulated (green) and down-regulated (red) by anti-IL-20 treatment arranged in order of fold modulation (increased on top and decreased on bottom) compared for each individual anti-IL-20 antibody treated animal compared to the vehicle treated animals. Three pancreatic tissues were evaluated in each group. Statistical evaluation is performed with 1-way ANOVA.

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induction of beta-cell apoptosis and promote insulin resistance [15]. The Psammomys obesus expressed both TLR2 and TLR4 in the myeloid cell compartment in the spleen. Interestingly, neutralization of IL-20 down-regulated the TLR4 expression specifically as the surface expression of TLR2 remained similar also after treatment. This suggests that IL20 signaling may be associated with pathways regulating the TLR4 expression and hence to amplify responses to endotoxins like the LPS. Indeed in dermatitis IL-20 has been shown to modulate the expression pattern of TLR4 towards the topical side in the upper layer of the affected skin has been demonstrated [51]. Further, IL-20 stimulates keratinocytes to produce the endogenous TLR4 agonist [52,53], which further increases the surface expression of TLR4 [54]. Thus, neutralization of IL-20 may thus result in reduced production of these endogenous TLR ligands that leads to reduced surface expression of TLR4. In obesity an accumulation of macrophages in the adipose tissue occurs which contribute to apoptosis of adipocytes and thereby to accumulation of lipids [55]. The crown-like structures of macrophages surrounding the dying adipocytes are highly responsive to TLR4 signaling [56]. The accumulation of the macrophages in the adipose tissue is regulated by several chemokines where the MCP-1 - CCR2b interaction is considered pivotal for pathogenesis [57]. Our demonstration that neutralizing anti-IL-20 down-regulate the expression levels of CCR2b on F4/80 macrophages hence suggests that the migratory capacity of the monocytes through this axis might be reduced. Further, given the reduced TLR4 expression after neutralization of IL-20, the relative reduction of weight gain rate in the treated Psammomys obesus might hence reflect reduced migratory capacity and modulated responsiveness of the macrophages by treatment. In metabolic disorders like obesity and diabetes, a complex interplay between proteins has been shown to influence disease progression dependent on when and where the protein is regulated. Of great interest, in the anti-IL-20 treated Psammomys obesus pancreas SDF-1/CXCL12 was highly significantly upregulated. CXCL12 has previously elegantly been shown by Yano et al. to attenuate diabetes by protecting β-cells from apoptosis [58].This is mediated by release of SDF-1 from the microvasculature in the Langerhans islets that acts on the CXCR4 expressing β-cells that responds through activation of AKT-signaling promoting cell survival. In the systemic compartment elevated SDF-1 is in contrast associated with disease and can in fact be used as a biomarker in blood to identify patients [59]. In line with our previous study in the diabetic db/db mouse, the tissue remodeling protein TIMP-1 is markedly upregulated in the anti-IL-20 treated pancreatic tissue [24]. Similar as SDF-1, local expression of TIMP-1 in the β-cells has been shown to protect from apoptosis and hence protect from diabetes [60]. Further, anti-IL20 treatment has been shown to promote hepatocyte proliferation and prevent liver fibrosis in tetrachloride induced liver injury [61]. Neutralization of IL-20 markedly down-regulates pro-inflammatory cytokines and chemokines like IL-1β, MIP1α and CXCL4 known to contribute to the low-grade inflammation in metabolic diseases both in the systemic compartment and in the pancreatic tissue. This anti-inflammatory effect by anti-IL-20 treatment is in line with our recent demonstration in the diabetic db/db mouse and hence strong suggests that IL-20 may play a significant role promoting low-grade inflammation [24]. With the recent demonstration that obesity and the development of obesity is tightly associated with pro-inflammatory responses and our demonstration herein that anti-IL-20 treatment reduces the pro-inflammatory responses and appears to potentially reduce the rate of weight gain suggests that direct modulation of inflammatory pathways during obesity development may indeed provide a novel mechanism to arrest the obesity progression. Taken together, our data shows for the first time presence and regulation of the immune system in the diabetic Psammomys obesus either by modulating leukocyte pre-cursor cell development of by interacting with IL-20R expressing structural cells or splenocytes. Importantly, this spontaneous diabetic model that has a normal NK population might prove useful to better understand the full spectrum of inflammation

during the development and progression of type 2 diabetes than the immune compromised db/db mouse model. This model might also prove useful in evaluation of the importance of inflammation during diabetic complications which currently is a great challenge due to lack of translation between pre-clinical models and clinical situation. Although the blood glucose and HbA1c was not modulated in response to neutralization of IL-20, the clear modulation of the low grade inflammatory response towards a phenotype with more MDSC and fewer M1-like macrophages suggests that neutralization of cytokines such as IL-20 might still provide beneficial therapeutic effects as an add-on therapy in obesity and maybe potentially also in T2D patients. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.intimp.2017.01.031. Author contributions H.C., L.H.T., A.R. researched the data and contributed to the discussion. H.C., L.H.T. critically read and commented on the manuscript. A.R. conceived the study, researched the data, contributed to the discussion and wrote the manuscript. A.R. is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors declare no competing interests. Acknowledgement We acknowledge the excellent technological expertise from Bodil Bosmann Jørgensen in conducting the protein arrays, cytokine evaluations and FACS analysis and Dr. Bidda Charlotte Rohlin for assistance with the in vivo studies and measurement. H.C. and L.H.T. received partial educational funds for this research. References [1] C.L. Hart, D.J. Hole, D.A. Lawlor, S.G. Davey, How many cases of type 2 diabetes mellitus are due to being overweight in middle age? Evidence from the Midspan prospective cohort studies using mention of diabetes mellitus on hospital discharge or death records, Diabet. Med. 24 (1) (2007 Jan) 73–80. [2] G.S. Meneilly, Pathophysiology of type 2 diabetes in the elderly, Clin. Geriatr. Med. 15 (2) (1999 May) 239–253. [3] E. Selvin, M.W. Steffes, C.M. Ballantyne, R.C. Hoogeveen, J. Coresh, F.L. Brancati, Racial differences in glycemic markers: a cross-sectional analysis of communitybased data, Ann. Intern. Med. 154 (5) (2011 Mar 1) 303–309. [4] Standards of medical care in diabetes, Diabetes Care 37 (Suppl. 1) (2014) S14–S80 (2014 Jan). [5] J.R. Greenfield, L.V. Campbell, Relationship between inflammation, insulin resistance and type 2 diabetes: ‘cause or effect’? Curr. Diabetes Rev. 2 (2) (2006 May) 195–211. [6] M.Y. Donath, Targeting inflammation in the treatment of type 2 diabetes: time to start, Nat. Rev. Drug Discov. 13 (6) (2014 Jun) 465–476. [7] G.S. Hotamisligil, Inflammation and metabolic disorders, Nature 444 (7121) (2006 Dec 14) 860–867. [8] M.E. Shaul, G. Bennett, K.J. Strissel, A.S. Greenberg, M.S. Obin, Dynamic, M2-like remodeling phenotypes of CD11c + adipose tissue macrophages during high-fat diet–induced obesity in mice, Diabetes 59 (5) (2010 May) 1171–1181. [9] L.H. Thomsen, Polarization of macrophages in metabolic diseases, J. Clin. Cell. Immunol. (2015) 6(2). [10] K. Fujiu, I. Manabe, R. Nagai, Renal collecting duct epithelial cells regulate inflammation in tubulointerstitial damage in mice, J. Clin. Invest. 121 (9) (2011 Sep) 3425–3441. [11] S.P. Weisberg, D. Hunter, R. Huber, J. Lemieux, S. Slaymaker, K. Vaddi, et al., CCR2 modulates inflammatory and metabolic effects of high-fat feeding, J. Clin. Invest. 116 (1) (2006 Jan) 115–124. [12] H. Cucak, L.G. Grunnet, A. Rosendahl, Accumulation of M1-like macrophages in type 2 diabetic islets is followed by a systemic shift in macrophage polarization, J. Leukoc. Biol. 95 (1) (2014 Jan) 149–160. [13] B. Calderon, A. Suri, X.O. Pan, J.C. Mills, E.R. Unanue, IFN-gamma-dependent regulatory circuits in immune inflammation highlighted in diabetes, J. Immunol. 181 (10) (2008 Nov 15) 6964–6974. [14] M. Boni-Schnetzler, J. Thorne, G. Parnaud, L. Marselli, J.A. Ehses, J. Kerr-Conte, et al., Increased interleukin (IL)-1beta messenger ribonucleic acid expression in beta -cells of individuals with type 2 diabetes and regulation of IL-1beta in human islets by glucose and autostimulation, J. Clin. Endocrinol. Metab. 93 (10) (2008 Oct) 4065–4074. [15] H. Cucak, C. Mayer, M. Tonnesen, L.H. Thomsen, L.G. Grunnet, A. Rosendahl, Macrophage contact dependent and independent TLR4 mechanisms induce beta-cell

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