PLOS ONE: Macrophage Contact Dependent and Independent TLR4 Mechanisms…

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Macrophage Contact Dependent and TLR4 Mechanisms Induce Dysfunction and Apoptosis in a Mouse of Type 2 Diabetes


2 diabetes (T2D) is evolving a global disease and patients a systemic low-grade inflammation, yet the of this inflammation is still not One plausible mechanism is enhanced and activity of the innate immune Therefore, we evaluated the expression and the of the toll-like receptor 4 (TLR4) on β-cells in primary mouse and on the murine β-cell line in the presence or absence of macrophages.

islets have 40% fewer positive β-cells, but twice the of TLR4 positive macrophages as to healthy islets. Healthy and islets respond to a TLR4 with enhanced production of (5–10-fold), while the TLR4 β-cell line MIN6 to produce cytokines. TLR4 induces β-cell dysfunction in islets, measured as reduced stimulated insulin secretion.

macrophages from 4-months old have acquired a transient capacity to produce cytokines stimulated with LPS. this is lost in 6-months old mice. TLR4 activation does not induce apoptosis in or MIN-6 cells.

In contrast, mediate TLR4-dependent cell-contact (3-fold) as well as cell-contact (2-fold) apoptosis of both and MIN-6 cells. Importantly, macrophages have a significantly capacity to induce β-cell compared to healthy macrophages. together, the TLR4 responsiveness is in the diabetic islets and mainly by newly recruited macrophages.

The positive macrophages, in both a dependent and independent manner, apoptosis of β-cells in a TLR4 fashion and TLR4 activation induces β-cell dysfunction. targeting either the TLR4 or the macrophages provides a novel treatment regime for T2D.

Cucak H, Mayer C, Tonnesen M, LH, Grunnet LG, et al. (2014) Macrophage Dependent and Independent TLR4 Induce β-Cell Dysfunction and in a Mouse Model of Type 2 PLoS ONE 9(3): e90685.

Editor: Christina Bursill, Research Institute, Australia

September 4, 2013; Accepted: 3, 2014; Published: March 3,

Copyright: © 2014 Cucak et al. is an open-access article distributed the terms of the Creative Commons License. which permits use, distribution, and reproduction in any provided the original author and are credited.

Funding: The study was through a post-doc grant the NovoNordisk foundation. The funders had no in study design, data and analysis, decision to publish, or of the manuscript.

Competing interests: The authors declared that no competing exist.


The global of Type 2 diabetes (T2D), to the rising obesity rate is to be one of the major public health of the 21st century and the fifth cause of … worldwide [2] .

T2D is characterized by defective insulin from the pancreatic β-cells and insulin sensitivity in peripheral leading to hyperglycemia [2]. The overload in these patients a low grade chronic inflammation in a progressive loss of pancreatic [3]. There is an intimate between the immune and metabolic systems with overlapping and inflammatory signaling and sensing

Factors such as lipids and provide cross-talk between and metabolic signaling pathways contribute to the risk of developing For example, overproduction of IL-1β in tissue is an important feature of and contributes significantly to insulin [4].

In human and animal elevated free fatty (FFA) levels have observed in obesity and T2D [4]. [5] toll-like receptors (TLRs) are to participate in sensing extracellular FFA TLRs are a family of pattern-recognition that play a critical in the innate immune system by pro-inflammatory signaling pathways in to microbial products [6].

is a subclass of TLRs that can be by lipopolysaccharide (LPS) and by nonbacterial such as saturated fatty [7]. TLR4 signals via two pathways, a MyD88 dependent or a independent pathway. The Myd88 pathway activates the transcription NFκB that in turn to the transcription of several pro-inflammatory resulting in the production of different cytokines such as IL-6, and TNFα.

The activation of TLR4 by and subsequent upregulation of intracellular pathways establish a link the innate immunity and diet obesity and insulin resistance. numerous studies consistently that TLR4 deficiency against the development of diet-induced and insulin resistance [7] –[9] .

are a heterogeneous population of cells various general markers, as F4/80, CD68, CD11b, and in a heterogeneous fashion, dependent on of origin, maturation, and activation [18]. The local tissue provides macrophages with stimuli creating an array of activated cells. Particularly, in the of Langerhans two distinct macrophage based on the expression of CD68 and are present.

The CD68 + F4/80 − macrophage invades the diabetic islets and an M1-like phenotype characterized by levels of CD11c over the marker CD206 [18]. The of the splenic cytokines in response to the glucose in early and late are macrophage-derived where the expanded + F4/80 − subpopulation is the predominant The diabetic macrophages acquire an capacity to synthesis pro-inflammatory as well as tissue remodeling such as TGF-β [18] .

if TLRs are mainly expressed on presenting cell such as and dendritic cells, TLR4 has identified on several other types including pancreatic [10]. as well as other sensitive tissues such as and muscle cells [7]. Whereas the role of TLR4 in insulin resistance is a well-established the specific roles of TLR4 cells in respect to the pathogenesis of T2D are an unexplored area.

Until TLR4 expression in islets has been examined under conditions and not with respect to development. However, a study by et al showed increased expression of mRNA accompanied by pro-inflammatory release in islets with the of T2D [12]. Whether this milieu might originate from the β-cells themselves, macrophages [13] or other in the islets is still an open

To assess the basis of TLR4 in the pathogenesis of T2D, we compared the mediated pro-inflammatory response of and spleen in diabetic db/db and mice. This mouse has a defective leptin receptor and significant obesity spontaneously. the mice also progress classical signs of T2D such as dysfunction, elevated blood and HbA1c as well as severe resistance [14].

This is in to the many commonly used induced obesity models all lead to obesity and moderately blood glucose, but never the classical characteristics of T2D [15]. Further, since inflammation β-cell apoptosis has been as a central component of the development and of T2D [17]. we explored the ability of and non-diabetic TLR4 activated to induce β-cell apoptosis.

Our suggests that the TLR4 pathways are indeed present and in the islets of Langerhans. The pathophysiological of the enhanced TLR4 activity in T2D to be enhanced induction of β-cell mediated by both direct contact dependent and independent

Materials and Methods

All animal were approved by the Copenhagen Ethics Committee and performed to their recommendations.

Male diabetic (db/db) and non-diabetic littermates (db/+) on C57BL/Ks were purchased from at 6 weeks of age and housed according to rules at the Novo Nordisk A/S facility (Måløv, Denmark). were given free to NIH-31 M rodent diet Laboratories, Boxmeer, Netherlands) and tap and maintained at 22–24°C.

Cell Reagents

Mouse MIN6 β-cells were cultured in (25 mM glucose, 2 mM l-glutamine, and 1 mM sodium supplemented with 10% FBS and 100 µM β-mercaptoethanol at and 5% CO 2 . LPS used was purchased from (L5543, Lot: 110M4090). Due to in LPS activity from batch/LOT full dose-response evaluations in cell type was performed and a specific concentration equivalent to an EC 80 of LPS was used in all functional experiments.

fatty acid (FFA) is (Sigma P5585) in a 50% solution Oleic acid (Sigma 50 mM solution of palmitate in 90% ethanol was to 60°C. Oleate was dissolved in 90% and heated to 60°C.

They mixed in a 1:1 solution, vortexed and into the right concentrations before use.

Isolation of Islets and Spleens and Generation of Cell Suspensions

8 week, 16 and 24 old db/db and db/+ mice … by cervical dislocation and and spleen were removed. For isolation, pancreas from 8 old mice was transferred to cold (grade II 300 units/ml, MP Biomedical), in the Grant/Edmund S25 thermoshaker at 200 strokes/min at for 10 min, transferred to a fresh with 75 units/ml collagenase, for an additional 5 min and the supernatants were The procedure was repeated several

Finally, the supernatants were three times in HBSS with 10 mmol/l HEPES, 5 NaHCO3, 1% P/S and 0.5% fetal serum (FCS) and allowed to for 5 min between each wash, and left on ice before hand-picking.

NMRI mice (Taconic) at weeks of age were … by dislocation. Liberase mix (117 Liberase TL (Roche); 25 mM CaCl2 14 units/µL DNaseI (BioNordika) in HBSS Ca2+/Mg2+ (Gibco) was into the pancreas via the common duct. The pancreas was dissected by digestion at 37°C for 18 min. activity was stopped with buffer (3 g/l BSA (Sigma-Aldrich); 0.5 g/l D-glucose diluted in HBSS Ca2+/Mg2+

The digest was filtered through a 400 µM strainer and after three islets were hand-picked purified islet fractions with 100 and 70 µM cell strainers.

islets were cultured in RPMI 1640 (RPMI + (Gibco) added 10% (vol/vol) FBS 1% (vol/vol) penicillin/streptomycin (Gibco), 20 mM (Gibco)) in a 5% CO 2 humidified atmosphere at

A single cell suspension the islets were generated by in trypsin-EDTA in HBSS w/o Ca 2+ /Mg 2+ at 37°C and up and down for 3 minutes followed by with FACS buffer 10% FCS, 2 mM EDTA).

Single suspensions were generated by spleens from db/db and mice through a 70 µm cell after which the erythrocytes lysed with cell buffer.

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Cytokine Production Islets

The isolated islets rested overnight in complete media and cultured in 96-well at concentration of 10 islets/well in the absence or of LPS (0.1 µg/ml, 1 µg/ml and 10 After 24 h, culture supernatants collected and analysed for IL-6, KC, RANTES and VEGF on the Bio-Plex 200 using the Milliplex map kit (Mouse magnetic bead panel) according to the manufacturer’s description.

Insulin Secretion in NMRI

Thirty islets per well transferred to an extracellular-matrix-coated 96-well (Biological Industries) in complete and incubated overnight in a 5% CO 2 humidified at 37°C. Medium was removed and islets were washed in (115 mM NaCl; 4.7 mM KCl; 2.6 mM 2 ; 1.2 mM KH 2 PO 4 ; 1.2 mM MgSO 4 ; 5 mM NaHCO 3 (all Merck); 1% penicillin/streptomycin (vol/vol) 20 mM Hepes (Gibco); 0.02 mM (Gibco); 0.2% (wt/vol) BSA pH 7.4).

Islets were incubated in KRBH added 3 mM for 0.5+1 h and KRBH added 15 mM with/without LPS or 150 pg/ml IL-1β and 5 IFNγ for 1 h. Buffer was collected the last 3 mM and 15 mM incubations, filtered a 96-well filter plate Millipore) and stored at −20°C analysis. Each experiment was to the insulin release at 15 mM ( = 100%). when calculating the average the 3 independent experiments, the variation of the 15 mM will be 0.15 mM glucose to the lowest concentration of glucose maximum insulin release.

Detection of insulin was by luminescence channeling immunoassay (LOCI). mAb RDI-TRK2IP10-D6C4 was conjugated to LOCI beads (PerkinElmer) and another mAb RDI-TRK2IP10-D3E7 (binding to a different was biotinylated. The assay was conducted in plates by adding 1 µl of calibrator, and unknown sample in the wells by 15 µl of a mixture of acceptor beads and antibody.

After 1 h of incubation at 30 µl of streptavidin-coated donor beads added and the plates were incubated for 30 min. The plates read in an Envision plate (PerkinElmer) at 21–22°C, applying a nm filter after excitation by a 680 nm The total measurement time per was 210 ms including a 70 ms excitation time. the assay the three reactants with analyte to form a complex.

Illumination of the complex chemiluminescence from the acceptorbeads is measured in the EnVision plate The amount of light generated is to the concentration of insulin. The concentration of was calculated against a standard of rat insulin using a 5 parameter

The lower limit of quantification was ng/ml.

Cytokine Detection

Cytokine production from islets or MIN6 cells was by treating 10 islets or 30 000 MIN6 in 96-well plates in a final of 100 µl. For quantification of cytokine production in 500 000 cells were treated in 400 µl in plates. Cells or islets treated with LPS for 24 h and supernatants assayed for IL-6, MCP-1, and IL-1β by AlphaLISA (Perkin-Elmer, MA) according to the manufacturer’s protocols.

were performed in triplicates.

of Cell Death

Apoptotic … in whole murine was determined in duplicates of size-matched by the detection of DNA-histone complexes in the cytoplasmic fraction of cells Cell Death Detection PLUS (Roche, Basel, Briefly, 10 islets were in 200 µl lysis buffer for 30 min at room

Lysates were centrifuged for 10 min at and 20 µl of the supernatant and 80 µl immuno-reagent (anti-DNA-POD and anti-histone-biotin antibody) were to streptavidin-coated microtiter plates and for 2 h under shaking conditions rpm) at room temperature. washing three times 200 µl incubation buffer, 100 µl ABTS was added to each well. was measured at 405 and 490 nm.

Apoptotic cell … in cells was assayed using the Triplex Assay kit from (Madison, WI). In brief, 30 000 cells were seeded in a volume of 100 µl 96-well plates and either treated after 24 h or or in transwell systems with at a ratio of 1:10 for an additional 24 h further treatment.

The cells then treated with 10 and 100 LPS, 150 pg/ml IL-1β and 5 IFNγ or conditioned media LPS-activated splenocytes (only for cultured MIN6). After 24 h of Caspase-Glo 3/7 Reagent was added to the and after 30 min of incubation at room luminescence was measured using the M4 (Molecular Devices). Quantification of 3/7 activity was measured in duplicates.

Cytometry Analysis

Flow analysis was performed according to procedures. Briefly, cells islet cells or splenocytes) first blocked for unspecific with anti-CD16/CD32 (BD Pharmingen) by surface staining of F4/80 and (Biolegend). Cells were fixed and permeabilized using Fixation/Permeabilization Solution Kit (BD Pharmingen) to manufacturer’s description and then stained for CD68 (AbD and insulin (RD Systems).

Intracellular levels were evaluated in cultured for 6 h at 37°C at 10 6 cells/ml in of 10 µg/ml BrefA and 100 ng/ml Cells were then for surface antigens including followed by fixation and permeabilization and stained for TNFα (eBioscience), (Biolegend), IL-1β (e-Bioscience), and Samples were then on a FACSFortessa equipped with red and violet laser followed by analysis using FACSdiva (BD Biosciences).

Statistical Analysis

analyses were performed GraphPad Prism 6 using two-tailed Student’s t-test or ANOVA with a multiple-comparisons

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