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0Cell Reports
Article
An MHC II-Dependent Activation Loop between Adipose Tissue Macrophages and CD4+ T Cells Controls Obesity-Induced Inflammation
Authors
Kae Won Cho, David L. Morris.....
Robert W. O'Rourke, Carey N. Lumeng
Correspondence
clumeng@umich.edu
In Brief
Obesity triggers an innate and adaptive immune response in adipose tissue, but little is known about how these signals are coordinated. Cho et al. demonstrate the importance of MHC II class-restricted signals from adipose tissue macrophages in controlling obesity-induced T cell activation and the development of insulin resistance. Loss of MHC II from resident tissue macrophages is sufficient to prevent the generation of active effector/ memory T cells identifying a target for intervention in metainflammation.
Highlights
Adipose tissue T cells dynamically interact with ATMs
MHCII in ATMs is required to generate effector/memory T cells with obesity
MHC II in resident ATMs is required for obesity-induced insulin resistance
Ablation of CD11c+ ATMs in obesity attenuates conventional T cell proliferation
Graphical Abstract
Cho et al., 2014, Cell Reports 9, 605-617 ciossMark October 23, 2014 ©2014 The Authors
http://dx.d0i.0rg/l 0.1016/j.celrep.2014.09.004
CelPress
Cell Reports
Article
An MHC II-Dependent Activation Loop between Adipose Tissue Macrophages and CD4+ T Cells Controls Obesity-Induced Inflammation
Kae Won Cho,16 David L. Morris,1 Jennifer L. DelProposto,1 Lynn Geletka,1 Brian Zamarron,2
Gabriel Martinez-Santibanez,1 Kevin A. Meyer,3 Kanakadurga Singer,1 Robert W. O'Rourke,34 and Carey N. Lumeng12 5 *
department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, MI 48109, USA 2Graduate Program in Immunology, University of Michigan Medical School, Ann Arbor, MI 48109, USA 3Department of Surgery, University of Michigan Medical School, Ann Arbor, MI 48109, USA 4Department of Surgery, Ann Arbor Veteran's Administration Hospital, Ann Arbor, MI 48109, USA 5Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI, 48109, USA 6Soonchunhyang Institute of Medi-bio Science, Soonchunhyang University, Cheonan-si, Chungcheongnam-do 330-930, South Korea 'Correspondence: clumeng@umich.edu http://dx.doi.org/10.1016/j.celrep.2014.09.004
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
SUMMARY
An adaptive immune response triggered by obesity is characterized by the activation of adipose tissue CD4+ T cells by unclear mechanisms. We have examined whether interactions between adipose tissue macrophages (ATMs) and CD4+ T cells contribute to adipose tissue metainflammation. Intravital microscopy identifies dynamic antigen-dependent interactions between ATMs and T cells in visceral fat. Mice deficient in major histocompatibility complex class II (MHC II) showed protection from diet-induced obesity. Deletion of MHC II expression in macrophages led to an adipose tissue-specific decrease in the effector/memory CD4+ T cells, attenuation of CD11c+ ATM accumulation, and improvement in glucose intolerance by increasing adipose tissue insulin sensitivity. Ablation experiments demonstrated that the maintenance of proliferating conventional T cells is dependent on signals from CD11c+ ATMs in obese mice. These studies demonstrate the importance of MHCII-restricted signals from ATMs that regulate adipose tissue T cell maturation and metainflammation.
INTRODUCTION
Obesity-induced adipose tissue inflammation is controlled by a diverse network of leukocytes comprised of multiple cellular regulators of innate and adaptive immunity (Mathis, 2013). One component of the metainflammatory response to obesity is an alteration in the state of adipose tissue T cells (ATTs) that influences the inflammatory set point of adipose tissue and insulin sensitivity. Adipose tissue contains a unique population of resident regulatory T cells (Treg) that is prominent in lean states and has a protective influence on adipose tissue inflammation in obesity (Cipolletta et al., 2012; Deiuliis et al., 2011; Feuerer
et al., 2009). While Tregs are downregulated with obesity, conventional Th1 T cells (Tconv) accumulate in visceral fat depots and contribute to proinflammatory signals in adipose tissue (Khan et al., 2014; Stolarczyk et al., 2013; Winer et al., 2009). The ability of Th1 ATTs to promote obesity-induced inflammation is dependent on ab T cell receptors (TCRs), T-bet, STAT3, and interferon g (IFNg) (Khan et al., 2014; O'Rourke et al., 2012; Priceman et al., 2013; Rocha et al., 2008; Stolarczyk et al., 2013). Th17 and Th22 cells in adipose tissue have also been associated with insulin resistance in obese individuals (Bertola et al., 2012; Fabbrini et al., 2013).
The signals that control the activation and maintenance of the ATTs are not well understood. Obesity induces ATT proliferation, suggesting that ATTs are stimulated by signals from the adipose tissue environment (Moraes-Vieira et al., 2014; Morris et al.,
2013). Both Treg and Tconv have a limited repertoire of TCRs, suggesting that clonal T cell selection shapes adipose tissue lymphocytes (Feuerer et al., 2009; Yang et al., 2010). Compared with secondary lymphoid tissues, adipose tissue contains few naive T cells and a high percentage of effector/memory type CD4+ T cells that regulate adaptive immunity based on interactions with antigen-presenting cells (APCs) (Reis e Sousa, 2006; Vandanmagsar et al., 2011; Yang et al., 2010; Zhu and Paul, 2008). APCs integrate multiple signaling pathways in tissues, resulting in the maturation of naive T cells toward a specific activation profile. For CD4+ T cells, a core component of the maturation process is the presentation of antigens via major histocompatibility complex II (MHC II), which binds to the TCR.
Several lines of evidence suggest that APCs partner with T cells in adipose tissue to control metainflammation. Global deficiency of MHC II protects mice from obesity-induced obesity and inflammation (Deng et al., 2013). Conversely, enhancing APC function in fat by injection of activated bone marrow-derived dendritic cells into mice promotes adipose tissue inflammation and induces insulin resistance (Moraes-Vieira et al., 2014; Stefanovic-Racic et al., 2012). Adipokines such as leptin, adipo-nectin, and RBP4 activate APCs and promote Th1 T cell activation (Jung et al., 2012; Mattioli et al., 2005; Moraes-Vieira et al.,
2014). APC signals also play a role in the maintenance of
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Figure 1. ATMs Physically Interact with CD4+ T Cells in an Antigen-Dependent Manner
(A) Immunofluorescence analysis of MHC II+ cells (red) In FALC regions of mouse adipose tissue.
(B) Immunofluorescence analysis of HLA-DR+(red) cells in human omental tissue. Representative images are presented from similar results from five independent samples.
(C) Maximum intensity projection of z stacks in FALCs in eWAT from CD11c-mCherry mice adoptively transferred with CFSE-CD4+ cells.
(D) Time-lapse images of interacting CD11c+ ATMs (red) and OTII CD4+ T cells (green) in eWAT from CD11c-mCherry mice injected with BSA (upper) or OVA (lower). Imaging of adipose tissue performed with intravital multiphoton confocal microscopy over a 20 min imaging window.
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protective adipose tissue Tregs. B7-deficient mice that lack cos-timulatory molecules CD80 and CD86 have reduced Tregs sys-temically and in adipose tissue and demonstrate worse adipose tissue inflammation (Zhong et al., 2014).
While APCs may shape ATTs, ATTs can also influence the recruitment and activation of adipose tissue macrophages (ATMs). ATT activation precedes the prominent accumulation of proinflammatory CD11c+ ATMs induced by chronic obesity (Nishimura et al., 2009; Winer et al., 2009). This observation implies that the machinery required to activate ATTs in response to obesity must be native to adipose tissue and exist prior to the onset of obesity. In all adipose depots in lean mice and humans, the most prominent professional APCs are the extensive network of MHC II+-resident ATMs (Lumeng et al., 2007; Odegaard and Chawla, 2011). Resident ATMs express markers of alternatively activated macrophages (CD206 and CD301/ MGL1) do not express the activation marker CD11c (CD11c~) and are concentrated in fat-associated lymphoid clusters (FALCs) and omental milky spots (Morris et al., 2013; Rangel-Moreno et al., 2009).
Work from our group and others has shown that ATMs are functional APCs that can promote antigen-specific T cell activation ex vivo and in adipose tissue (Bertola et al., 2012; Morris et al., 2013; Stefanovic-Racic et al., 2012). However, several unresolved questions remain regarding if MHC II-restricted signals from ATMs are required for the activation of CD4+ T cells in adipose tissue, the importance of MHC II signals in insulin resistance, and which subtype of ATM (resident or recruited CD11c+) is required for initiating an adaptive immune response to obesity. The goal of this study was to investigate the role that ATMs play in directing CD4+ ATT activation, adipose tissue inflammation, and insulin resistance in vivo. We reveal a dynamic and antigen-dependent network of immune surveillance driven by interactions between ATMs and ATTs in visceral adipose tissue in vivo. Interruption of communication between resident tissue ATMs and ATTs via macrophage-specific deletion of MHC II does not alter ATTs in lean states, but prevents the generation of effector/memory ATTs and adipose tissue insulin resistance with obesity. Ablation of CD11c+ cells in obese mice support the importance of CD11c+ ATMs in maintaining Tconv at the expense of Treg. Overall, our studies identify a critical MHC II-restricted activation loop between resident CD11c~ ATMs, ATTs, and CD11c+ ATMs that contributes to metainflammation.
RESULTS
CD4+ T Cells Demonstrate Antigen-Dependent Interactions with ATMs In Vivo
We have previously identified high concentrations of CD4+ ATTs in FALC structures in visceral adipose tissue that are also enriched for MHC II+ CX3CR1 +ATMs (Morris etal., 2013). To better delineate relationship between CD4+ T cells and ATMs in FALCs,
we performed confocal microscopy on whole-mount adipose tissue samples. FALCs contained Mgl1 +ATMs that coexpressed MHC II (Figure 1A). MHC II was not identified in caveolin+ adipocytes in lean or high-fat diet (HFD)-fed obese mice. Imaging of human omental adipose tissue from obese patients undergoing bariatric surgery identified milky spots enriched for CD206+ HLA-DR+ ATMs (Figure 1B). HLA-DR+ cells were in direct contact with HLA-DR~ CD4+ T cells in milky spots. As in mice, no HLA-DR staining was observed in association with adipocytes.
We employed live intravital microscopy to evaluate the dynamic interactions between ATMs and CD4+ ATTs in vivo. CFSE-labeled CD4+ T cells from OT-II mice were injected intraperitoneal (IP) into CD11c-mCherry reporter mice that permitted identification of endogenous myeloid cells (Khanna et al., 2010). Flow cytometry demonstrated that the mCherry+ cells were F4/ 80+ CD11b+ ATMs in lean mice (Figure S1A). At 24 hr after injection, recipient mice were anesthetized, and epididymal white adipose tissue (eWAT) was externalized for imaging. More than 20% CD4+ T cells isolated from eWAT were CFSE+ (-15% of injected cells were recovered in adipose tissue) and concentrated in FALC structures (Figures 1C, S1B, and S1C). Cells were imaged by multiphoton confocal microscopy for 20 min. While mCherry+ cells did not demonstrate significant motility, the CD4+ T cells were highly motile (mean velocity = 5.6 mm/min) and showed random movement within the FALC structure (Figures 1D and 1E; Movie S1).
The interactions between CD4+ T cells and APCs are increased in the presence of a cognate T cell-specific antigen (Halin et al., 2005; Koltsova et al., 2012). To examine this in adipose tissue, CD11c-mCherry mice were injected IP with CFSE-labeled CD4+ OT-II T cells followed by injection of ovalbumin (OVA) or BSA control. After 2 hr, CD4+ T cell velocity and path of movement were assessed with intravital microscopy (Figures 1F and 1G). In BSA-injected mice, the velocity of CD4+ T cells and displacement length were 5.53 ± 0.36 mm/min and 25.14 ± 0.52 mm, respectively, over 20 min. In control mice, less than 20% of the T cells directly interacted with mCherry+ cells based on colocalization (Figure 1H). Among the motile CD4+ cells, -2% of the cells showed distinct high-velocity motile behavior. With OVA injection, long-lived T cell-CD11c+ cells interactions were induced, and -60% of the CD4+ T cells maintained stable contact with the mCherry+ cells over 20 min (Figure 1H; Movie S2). With OVA, the average speed (2.2 ± 0.14 mm/min) and mean displacement length (BSA versus OVA: 25.15 ± 0.52 versus 14.73 ± 1.2 mm; p < 0.05) of CD4+ T cells were decreased compared with controls. The reduction in migration velocity with OVA was due to a reduction in mean individual T cell motility as well as the loss of the high-motilityT cells seen in control mice. In summary, intravital imaging demonstrates that dynamic interactions between ATMs and CD4+ T cells in adipose tissue are constantly occurring in adipose tissue and such interactions are stabilized and enhanced in an antigen-dependent manner.
(E) CD4+ T cell migration pathways In adipose tissue from mice Injected with BSA (upper) or OVA (lower). (F and G) Velocity (F) and displacement lengths (G) of individual CD4+ T cells in eWAT.
(H) Percentage of CD4+ T cells interacting with CD11c+ cells in eWAT. Microscopy data are representative of three independent experiments. Data are means ± SEM. **p < 0.01, ***p < 0.001 versus BSA.
Figure 2. MHC II-Deficient Mice Are Protected from HFD-Induced Insulin Resistance through the Reduction of ATMs and CD4+ ATT Accumulation
MHC IIKO (KO) and WT control male mice were fed ND or HFD for 20 weeks.
(A) Body weights of WT and MHC IIKO (KO) mice during ND and HFD feeding (ND, n = 6; HFD, n = 8).
(B) Body composition analysis of fat and lean mass in WT and KO mice as determined by NMR. The right panel shows normalization to total body weight.
(C) Organ weights at the end of the diet exposure.
(D) Immunofluorescence identification of Mac2+ ATMs in eWAT from HFD-fed WT and KO mice. Scale bar represents 200 mm.
(E) Adipocyte size and adipocyte size distribution in eWAT from HFD-fed WT and KO mice. (F and G) Analysis of glucose metabolism by (F) GTTs and (G) ITTs.
(H) Quantitation of total, CD11c+, and CD11c" ATMs (CD11b+F480+) in eWAT from WT and KO mice by flow cytometry.
(I) Quantitation of CD3+ CD4+ and CD3+ CD8+ ATTs (left), Tconv and Treg (right) in eWAT in WT and KO mice. (J) Quantitation of blood CD3+ CD4+ and CD3+ CD8+ T lymphocytes from WT and KO mice.
Data are shown as means ± SEM; #p < 0.05 in one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001 versus WT.
MHC II Is Required for HFD-Induced Adipose Tissue Inflammation and Insulin Resistance
Given that MHC II is required for cell-mediated immunity through CD4+ T cells, we examined the requirement for MHC II in the generation of obesity-induced inflammation by studying MHC II-deficient mice (H2-Ab1-/-, MHC II KO). Lean MHC II KO mice weighed less then age-matched wild-type (WT) mice and gained less weight with age (Figure 2A). When challenged with a HFD for 20 weeks, MHC II KO mice gained weight at a reduced rate compared with WT controls (WT 27.8 ± 0.6 g; MHC II KO 20.6 ± 1.2 g; p < 0.01). Metabolic cage analysis did not identify any differences in food intake, energy expenditure, or body length between genotypes in either diet condition (Figure S2). A body composition analysis showed that the weight differences between genotypes were due to a decrease in lean body mass, but not fat mass, in normal diet (ND) and HFD MHC II KO mice (Figure 2B). When body composition was expressed as a percentage of total body mass, there were no differences between genotypes in ND and HFD condi-
tions. Tissue weights at sacrifice demonstrated significantly more visceral adiposity in HFD-fed MHC II KO compared with WT (Figure 2C). HFD-fed WT and MHC II KO mice demonstrated similar hypertrophy of inguinal/subcutaneous fat depots (sWAT). HFD-fed mice demonstrated a significant accumulation of Mac2+ crown-like structures (CLSs) in WT eWAT, while CLSs were largely absent in MHC II KO adipose tissue, consistent with a preservation of adipose tissue health and low inflammation (Figure 2D). Adipocyte size did not differ between HFD-fed WT and MHC II KO mice. (Figure 2E). Liver weights of MHC II KO mice were significantly less than WT mice in both dietary conditions, and a HFD did not induce an increase in liver weight in MHC II KO mice (Figure 2C). HFD MHC II KO mice were protected from histologic steatosis and liver triglyceride accumulation (Figure S3A). This protection was associated with a significant decrease in hepatic Scd1 and Fasn expression in MHC II KO mice (Figure S3D).
Glucose and insulin tolerance were not altered in ND-fed MHC II KO mice (Figures 2F and 2G). Despite an increase in
visceral adipose tissue, obese MHC II KO mice had improved glucose and insulin tolerance compared with HFD WT mice. Insulin levels and free fatty acids were significantly lower in obese MHC II KO mice compared with WT (Figure S3B). Gene expression analysis of eWAT from HFD mice showed increased expression of genes associated with adipocyte insulin sensitivity (e.g., Glut4, Pparg, and Adipoq) in MHC II KO mice compared with WT (Figure S3C). In addition, MHC II KO mice showed decreased expression of Ifng in eWAT, indicating that MHC II-restricted signals are required for maximal Ifng induction with obesity. In liver, there were no significant differences in Ifng, Tnfa, Il6, or Foxp3 gene expression between WT and MHC II KO mice in response to HFD (Figure S3D). Overall, these data demonstrate that MHC II-dependent signals contribute to insulin resistance with diet-induced obesity by promoting adipose tissue dysfunction and hepatic lipid accumulation.
MHC II Deficiency Reduces the Accumulation of CD11c+ ATMs and CD4+ ATTs, but Not CD8+ ATTs, in Response to HFD
To examine the effects of MHC II on leukocyte infiltration into visceral fat, ATM and ATT content was assessed by flow cytom-etry in eWAT from WT or MHC II KO mice. Consistent with the imaging data, MHC II KO mice had fewer ATMs due to a decrease in the percentage of CD11c+ ATMs (Figure 2H). The reduction in CD11c+ ATM accumulation could not be explained by reduced MCP-1 or reduced circulating monocyte levels, as Ccl2 expression in eWAT and the content of 7/4hi blood monocytes were similar between HFD-fed WT and MHC II KO mice (Figures S3C and S3E). Consistent with the importance of MHC II in thymic selection of CD4+ T cells, MHC II KO mice had a significant reduction in CD4+ lymphocytes in the blood, spleen, and adipose tissue compared with WT mice in both diet conditions (Figures 2I and 2J; data not shown). A substantial reduction of CD4+ ATTs as a percent of stromal vascular cells (SVCs) was seen for both Tconv (CD4+ FoxP3~) as well as Tregs (CD4+ FoxP3+). CD8+ T cells were significantly increased systemically and in adipose tissue of MHC II KO mice. These results suggest that neither an increase in CD8+ ATTs nor a reduction in Tregs alone is sufficient to generate maximal adipose tissue inflammation and insulin resistance and suggest an important role for MHC II-dependent Tconv in obesity-induced inflammation.
Macrophage-Specific MHC II-Deficient Mice Show Improved Insulin Sensitivity in Response to HFD
The systemic depletion of CD4+ cells in whole-body MHC II KO mice does not let us discern the role of adipose tissue-specific MHC II in regulating T cells. To address this, initial experiments attempted to delete MHC II expression in hematopoietic cells via bone marrow transplantation of MHC Ir/_ donors into WT recipients. However, similar to other reports (Marguerat et al., 1999), these mice developed an autoimmune phenotype with severe weight loss that limited our ability to assess metabolic responses (data not shown).
Resident CD301+ ATMs are the dominant professional APC in adipose tissue by quantity in lean states and are thus poised to initiate T cell activation in response to obesogenic signals
(Bertola et al., 2012; Stefanovic-Racic et al., 2012). To evaluate the hypothesis that MHC II signals from resident ATMs are required for obesity-induced activation of CD4+ ATTs, we generated macrophage-specific MHC II knockout (MMKO) mice. LysM-Cre mice were crossed to mice with flox sequences flanking exon 1 of the H2-ab1 gene (H2-Ab1tm1Koni; MHC IIf/f). Flow cytometry confirmed MHC II expression in both ATMs and adipose tissue dendritic cells (ATDCs) in MHC IIf/f control mice. In this experiment, the macrophage-specific marker CD64 was used to identify ATMs (resident CD45+ CD64+ CD11c~ and recruited CD45+ CD64+ CD11c+) and distinguish them from ATDCs (CD45+ CD64~ CD11c+) (Gautier et al., 2012) (Figure 3A). Lean MMKO did not demonstrate any differences in the quantity of ATMs or ATDCs in eWAT compared with controls (Figure 3B). However, MHC II expression was absent in >95% of the ATMs of MMKO mice and remained intact in a small but prominent population of ATDCs. Immunostaining of adipose tissue confirmed deletion of MHC II in MGL1+ resident ATMs in MMKO mice (Figure 3C). Expression of MHCII (H2-ab1) in eWAT was decreased by >95% in MMKO mice without changing Emr1, CD40, or CD80 expression demonstrating that LysM-cre expressing cells account for the vast majority of MHCII expression in adipose tissue (Figure 3D). MMKO mice had normal numbers of circulating neutrophils and monocytes, indicating that the macrophage MHC II is not required for the proper maintenance of myeloid cells (Figure 3E). Importantly, there were no changes in CD4+ or CD8+ T cells in the thymus, spleen, and lymph nodes of MMKO mice, demonstrating that macrophage MHC II is dispensable for normal T cell maturation in lymphoid tissues (Figure 3F).
To determine the importance of macrophage MHC II in glucose metabolism, control and MMKO mice were fed with ND or HFD for 14 weeks. MMKO mice were healthy and demonstrated weight gain with HFD that was identical to control mice (Figure 4A), suggesting that macrophage MHC II does not account for the growth defects observed in whole-body MHC IIKO mice. eWAT hypertrophy on HFD was similar between control and MMKO mice (Figure 4B). Fasting glucose and insulin levels did not differ between ND-fed control and MMKO mice (Figure 4C). HFD-fed MMKO mice showed a significant decrease in fasting glucose levels compared with control mice, although fasting insulin levels were similar. Glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) did not differ between ND-fed control and MMKO mice (Figures 4D and 4E). However, when challenged with HFD, MMKO mice had significantly improved glucose tolerance and insulin sensitivity compared with controls.
Immunofluorescence microscopy showed a reduction in CLSs in eWAT from obese MMKO mice compared with controls without differences in adipocyte size (Figures 4F and 4G). To evaluate the mechanisms of improved insulin sensitivity in MMKO mice, adipose tissue and liver insulin signal trans-duction was evaluated in obese mice. Consistent with the decrease in CLSs, HFD-fed MMKO mice demonstrated a significant increase in Akt phosphorylation in eWAT in response to insulin compared with WT mice (Figure 4H). Insulin-stimulated Akt phosphorylation in the liver was marginally increased in MMKO mice compared with controls (Figure 4I). Gene
Figure 3. Generation of Macrophage-Specific MMKO Mice
(A) Representative flow cytometry plots of MHC II-expressing cells In eWAT SVCs. ATMs (CD45+ CD64+) (upper panels) and ATDCs (CD45+ CD64~ CD11c+) (lower) from control (CON) and MMKO mice.
(B) Quantitation of ATMs and ATDCs (upper) and frequency of MHC II+cells in ATMs and ATDCs (lower) from CON and MMKO mice. Five mice were assessed per group in four independent cohorts.
(C) Immunofluorescence images showing loss of MHC II+ expression in (green) and resident Mgl1 + ATMs (red). Scale bar represents 50 mm.
(D) Quantitative PCR analysis of eWAT from CON and MMKO mice.
(E) Quantitation of total blood myeloid cells and total lymphocytes.
(F) Frequency of CD4+ and CD8+ T lymphocytes in thymus, spleen, and mesenteric lymph nodes (mLNs). Data are means ± SEM. ***p < 0.001 versus CON.
expression from whole adipose tissue demonstrated a significant increase in adiponectin (Adipoq) and Glut4 expression in EWAT from obese MMKO mice compared with WT (Figure 4J). These observations indicate that MMKO mice are protected from insulin resistance by improvements in adipose tissue insulin sensitivity.
Accumulation of CD11c+ ATMs in Visceral Fat Is Attenuated in MMKO Mice
To examine the cellular mechanisms involved in the improved metabolism of MMKO mice, ATM and ATDC content was analyzed by flow cytometry (Figure 5A). There were no significant differences in total ATMs or ATDCs in eWAT between control
Figure 4. Macrophage-Specific MHC II Knockout Mice Have Attenuated HFD-Induced Insulin Resistance due to Improved Adipose Tissue Insulin Sensitivity
Metabolic assessment of MMKO and control mice fed ND or HFD for 14 weeks (n = 10-12 for each group).
(A) Body weight.
(B) eWAT weight.
(C) Fasting glucose (left) and insulin levels (right).
(D and E) GTT (D) and ITT (E) performed at 12 weeks of diet exposure.
(F) Immunofluorescence image of Mac2+ ATM (red) and caveolin+ adipocytes (green) in eWAT from HFD fed CON and MMKO mice. Scale bar represents 100 mm.
(G) Quantitation of adipocyte size in eWAT from HFD-fed CON and MMKO. At 12 weeks after HFD feeding, tissues were harvested from CON and MMKO mice 5 min after intravenous injection of saline or insulin.
(H and I) Immunoblots of lysatesfrom eWAT(H) and liver (I) with anti-pSer473 or anti-Akt (upper) and quantitation of phosphorylated AKT normalized to total AKT levels (lower).
(J) Gene expression in eWAT from HFD fed CON and MMKO. Data are means ± SEM. *p < 0.05, **p < 0.01 versus CON.
and MMKO mice in lean or obese states (Figure 5B). In ND-fed mice, the quantity of CD11c+ and CD11c~ resident ATMs as a percentage of SVCs did not differ between genotypes. However, with HFD, obese MMKO mice had significantly fewer CD11c+ ATMs compared with controls. No changes in the quantity of resident CD11c~ ATMs were observed in MMKO mice, resulting in an overall decrease in the ratio of CD11c+ to CD11c~ ATMs in obese MMKO mice (Figure 5C). Consistent with this, the M1 macrophage marker Nos2 was decreased in eWAT, and the alternatively activated macrophage marker Arg1 showed a trend toward an increase (Figure 5D). To evaluate whether differences in proliferation account for the decrease in CD11c+ATMs, tissue sections were stained with Ki67. No differences in Ki67+ cells in
the ATM-enriched CLSs were observed (Figure 5E). Analysis of serum from obese mice identified a significant decrease in MCP-1 levels in MMKO mice compared with WT controls (Figure 5F). Bone marrow-derived macrophages were generated from WT and MMKO mice, and no differences in inflammatory gene expression in response to lipopolysaccharide (LPS) were observed, suggesting that MHC II is not required for macrophage activation (Figure 5G). In agreement with the lower MCP-1 and ATM accumulation, expression of genes associated with inflammation such as Tnfa and Il10 in eWAT and liver from obese MMKO was decreased compared with obese WT controls (Figures S4A and S4B). Collectively, these results indicate that the presence of MHC II on resident ATMs is required for maximal
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(A) Representative flow cytometry plots of CD45+ cells in SVC from eWAT from HFD-fed CON and MMKO. ATMs (CD45+ CD64+) are differentiated based on CD11c expression. ATDCs are defined as CD64~ CD11c+.
(B) Quantitation of ATM and ATDC content in eWAT.
(C) Frequency of CD11c+ ATMs and CD11c" ATMs (left) and the ratio of CD11c+ ATMs to CD11c" ATMs (right) in eWAT.
(D) Gene expression in eWAT from HFD-fed CON and MMKO.
(E) Quantitation of Ki67+ cells frequency in eWAT from HFD-fed CON and MMKO.
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The Activation of Conventional CD4+ ATT Is Dependent on Macrophage MHC II
We next examined the effect of MHC II deficiency in resident ATMs on ATTs. The total amount of CD3+ lymphocytes in eWAT was not significantly different between control and MMKO mice on a ND or HFD (Figure 6A). While the quantity of CD4+ ATTs was similar between lean WT and MMKO mice, obese MMKO mice had fewer CD4+ ATTs in eWAT compared with HFD controls (Figure 6B). In ND mice, there were no differences between MMKO and controls in the quantity of Tconv and Treg in eWAT (gating shown in Figure S5A). However, with HFD, Tconv expansion was blocked in MMKO mice (Figure 6C). In our cohorts, Tregs increased with HFD as a percentage of SVCs in control mice, but this was blunted in MMKO mice (Figure S5B). Despite the selective reduction in CD4+ ATT in MMKO mice, no significant differences in proliferating Ki-67+ CD4+ ATTs were observed in ND- or HFD-fed MMKO mice compared with controls (Figure 6D). Lean MMKO mice
demonstrated an increase in the number of CD8+ ATTs in eWAT (Figure 6B). With a HFD, CD8+ ATTs increased to a similar degree in control and MMKO mice.
Quantitation of naive, effector-memory (E/M) and central-memory (C/M) CD4+ T cell subsets demonstrated similar numbers of these subsets in ND fed MMKO and control mice (Figures 6E-6G), suggesting that homeostatic ATT proliferation is MHC II independent. HFD exposure decreased naive CD4+ ATTs and increased E/M CD4+ ATTs in eWAT in WT mice. In contrast, HFD-fed MMKO mice retained similar percentages of naive T cells, as ND mice had a decrease in the number of E/M ATTs. No significant changes in C/M ATTs were observed between genotypes in either diet condition (data not shown). These changes in CD4+ T cells were adipose tissue specific as no differences in the frequency of CD4+ T cell, CD8+ T cells, Tconv, Treg, and proliferation were observed in the spleens of obese control and MMKO mice (Figure S5C). Gene expression analyses identified slight reductions Il2, a critical regulator of T cell activation, in eWAT from MMKO mice (Figure S4A). No differences in transcription factors critical for Th1, Th2, and Th17 differentiation (Tbet, GATA3, RORg) or polarization signals (Il4) were seen in whole eWAT from obese WT and MMKO mice. Gene expression analysis of FACS purified CD4+ T cells from eWAT from MMKO mice showed a significant decrease in Ifng expression compared with WT (Figure 6H). This difference was specific to ATTs as Ifng expression in splenic CD4+ T cells did not differ between MMKO and WT mice (Figure S5C). These findings suggest that the maturation of CD4+ ATTs with chronic HFD is dependent on ATM-derived MHC II.
Figure 6. Macrophage-Specific MHC II Deficiency Reduces Obesity-Induced CD4+ ATT Accumulation and Maturation
(A and B) Quantitation of (A) CD3+ ATTs and (B) CD4+ and CD8+ ATTs in eWAT.
(C) Quantitation of Tconvs and Tregs in eWAT expressed as % SVCs (left) and as % CD4+ ATTs (right) in CON and MMKO.
(D) Frequency of proliferating Ki67+ CD4+ ATTs in eWAT.
(E) Representative flow cytometry plots gated on CD3+ CD4+ cells to quantify E/M (CD44high CD62L~), C/M (CD44high CD62L+) and naive (CD44low CD62L+) CD4+ ATT subsets in eWAT.
(F) Quantitation of naive CD4+ ATT subsets in eWAT.
(G) Quantitation of E/M CD4+ ATT subsets in eWAT.
(H) Ifng gene expression in FACS-sorted CD4+ ATTs from HFD-fed CON and MMKO. Data are means ± SEM. *p < 0.05, **p <0.01, ***p < 0.001 versus CON.
Ablation of CD11c+ Cells Reduces CD4+ ATT Accumulation and Proliferation in Obese Mice
The MMKO results demonstrate the importance of resident CD11c~ ATMs in initiating ATT activation in response to obesity. Since an increase in APC function is associated with the accumulation of CD11c+ ATMs in obese mice, we examined the importance of CD11c+ ATMs in sustaining T cell activation in established obesity using a CD11c+ cell ablation model. Chimeric CD11c-DTR mice were generated by transplanting bone marrow from CD11c-DTR transgenic mice (CD45.2) into lethally irradiated wild-type recipients (CD45.1) (Figure 7A). Six weeks after reconstitution, chimeric mice were fed a ND or HFD for 6 weeks. Mice were then injected IP with PBS or diphtheria toxin (DT) every other day for 2 weeks while continuing the diets. No significant changes in total body weight or eWAT mass were observed with DT injection (Figures S6A and S6B). Efficient ablation of CD11c+ ATMs was confirmed by flow cytometry (Figure 7B). As expected, in control mice, HFD increased total eWAT ATM content and induced MHC IIhigh CD11c+ ATMs (Figures 7C and 7D). DT injection led to a slight decrease in total ATMs in lean
mice and significantly decreased the number of ATMs in HFD mice. CD11c ablation did not alter fasting glucose levels or glucose tolerance in ND-fed mice but normalized fasting glucose levels and significantly improved glucose tolerance (Figures S6C and S6D).
We next examined the effect of CD11c+ ATM ablation on ATTs. In PBS controls, the quantity of CD3+ ATTs in eWAT increased in response to HFD (Figure 7E). Ablation of CD11c+ ATMs did not alter CD3+ ATTs in ND-fed mice, but led to a significant decrease in CD3+ ATTs in obese mice. This decrease was driven by a decrease in CD4+ T cells (Figure 7F). Depletion of CD11c+ ATMs in HFD mice decreased the number of Tconv, while Tregs increased in eWAT in the DT-treated group (Figure 7G). This resulted in a significant decrease in the ratio of Tconv to Tregs induced by with CD11c cell ablation in both diet groups. CD4+ ATT proliferation was assessed by BrDU incorporation. HFD increased the proliferation of Tconv in controls (Figures 7H-7J). Ablation of CD11c+ ATMs did not alter Tconv or Treg proliferation in ND mice. However, depletion of CD11c+ ATMs in HFD mice decreased Tconv proliferation and enhanced Treg proliferation
in adipose tissue. Gene expression analysis of eWAT showed that depletion of CD11c+ ATMs decreased expression of genes associated with T cell activation (Il2 and Il12) and increased expression of genes associated with Treg function (Foxp3, GATA3, and Il10) (Figure 7K). Overall, these data suggest that CD11c+ ATMs contribute to the maintenance Tconv ATTs at the expense of Tregs in obese mice.
DISCUSSION
Our studies suggest important roles for both resident CD11c~ and recruited CD11c+ ATMs as initiators and effectors of adipose tissue inflammation via their communication with ATTs via MHC II (Figure 7L). Chronic adipose tissue expansion leads to events that compromised adipocyte function (e.g., hypoxia, ER stress). MHC II+ resident ATMs (CD11c" MGL1/CD301 + in mice) reside in close proximity to adipocytes and sample the environment. ATTs transit throughout adipose tissue and FALCs/milky spots, where they interact transiently with MHC II+
Figure 7. Ablation of CD11c Decreases CD4+ ATT Accumulation and Proliferation in Response to Obesity
(A) Schematic diagram of experimental design of DT-mediated ablation of CD11c-expressing cells. Five mice per group were analyzed.
(B) Representative analysis of MHC II expression in ATMs (CD11b+F480+) in eWAT from PBS or DT-treated HFD-fed mice demonstrating loss of MHC IIhigh cells with DT.
(C-F) Quantification of (C) ATMs, (D) CD11c+MHC IIHI ATMs, (E) CD3+ ATTs, and (F) CD4+ ATTs in eWAT.
(G) Quantification of Tconv and Treg (left) and the ratio of Tconv to Treg (right) in eWAT.
(H) Representative analysis of CD4+ ATTs after BrdU injection to identify proliferating cells. Cells were gated on CD3+ CD4+ ATTs and then examined for FoxP3 and BrdU intracellular staining to assess Tconv (Foxp3~) and Treg (Foxp3+) ATT in HFD-fed mice.
(I and J) Quantitation of proliferating (I) Tconv and (J) Treg in eWAT.
(K) Quantitative RT-PCR analysis of eWAT from
PBS or DT-treated HFD mice.
Data are means ± SEM. *p < 0.05, **p < 0.01, ***p <
0.001 versus PBS.
(L) Graphical model.
ATMs. Interactions between ATTs and ATMs are enhanced by cognate antigens to potentiate T cell activation and conversion of naive to effector/memory T cells. MHC II in resident ATMs is required to translate obesogenic cues into a maturation of Tconv to this effector/memory phenotype with increased IFNg expression. Disruption of class II signals in resident ATMs prevents the downstream accumulation of proinflammatory CD11c+ ATMs and preserves adipocyte insulin sensitivity. Once established, CD11c+ ATMs support the continued activation and proliferation of Tconv at the expense of Tregs to promote insulin resistance.
Macrophage MHC II is not required for T cell maturation in primary and secondary lymphoid tissues, permitting us to identify adipose tissue specific effects. Deletion of MHC II in macrophages (MMKO mice) does not alter ATT development consistent with the MHC II-independent homeostatic proliferation of tissue memory T cells (Surh and Sprent, 2008). However, loss of macrophage class II restricted signals impairs their activation with obesity demonstrating the importance of ongoing T cell education and selection in adipose tissue. This is consistent with the unique profile of adipose tissue Tregs (Feuerer et al., 2009). Consistent with reports of a role for MHC II in shaping CD8 maturation (Doetal., 2012), both MMKO and MHC IIKO mice have an increase in adipose tissue CD8+ cells, suggesting that an increase in CD8+ T cells in fat alone is not sufficient to potentiate insulin resistance. Moreover, MHC IIKO mice have a significant
reduction in Tregs, yet are protected from obesity-induced inflammation. This finding seems to be at odds with other studies demonstrating that Tregs are the sole protective factor in adipose tissue that prevents inflammation. Our experience examining adipose tissue Tregs in lean and obese mice has differed from other reports, as we do not consistently see a decrease in adipose tissue Tregs with obesity as a percentage of CD4+ T cells. Because of the increase in total CD4+ T cells in the SVF with obesity, we frequently observe an increase in Tregs as a percentage of the SVF consistent with other reports (Deiuliis et al., 2011; Winer et al., 2009). We are unsure of why our results differ from other groups on this point, but differences in animal husbandry conditions may contribute to Treg variation.
Our results with macrophage-specific MHC II deletion and ablation of CD11c+ cells are complementary and reveal distinct in vivo functions of ATM subtypes in T cell activation. While the MMKO mice argue for a critical role for resident ATMs in the initiation of T cell activation with obesity, CD11c-DTR experiments suggest an important role for CD11c+ ATMs in sustaining this activated state. Similar to other reports, ablation of CD11c+ ATMs in obese animals reverses insulin resistance and adipose tissue inflammation (Patsouriset al., 2008). Our results link these improvements to a decrease in the number and proliferative capacity of Tconv and an increase in Tregs when CD11c+ ATMs are ablated. This demonstrates the importance of both ATM subtypes in regulating T cell activation. Resident ATMs participate in the initiation of inflammatory cascades via MHC II to induce CD11c+ ATMs. However, in an established obese inflammatory environment, CD11c+ ATMs play an important role in sustaining conventional T cell proliferation and suppressing Tregs.
Our data demonstrate a critical role ATMs play in priming CD4+ T cells and sustaining adaptive immune responses to obesity in vivo and are consistent with in vitro studies (Moraes-Vieira et al., 2014; Morris et al., 2013). Adipocytes have been shown to induce MHC II gene expression with obesity and reactivate antigen-primed T cells in vitro (Deng et al., 2013). Our studies have found little evidence to support significant MHC II protein expression on adipocytes in obese mice or human adipose tissue samples relative to ATMs. By gene expression, macrophages account for >95% of the MHC II expression in adipose tissue (residual MHC II+ adipose tissue DCs remain in our MMKO mice), arguing that nonmacrophage cells such as adipocytes play a minor role in MHC II expression in adipose tissue. Our studies show that CD4+ ATTs interact with ATM-derived MHC II much more frequently in their environment. However, it is clear that regulation of T cell homeostasis is not purely MHC II dependent, and it is likely that adipocyte derived signals influence T cell function and activation.
A major unanswered question in the field is the nature of the antigen that is being presented by ATMs and sensed by T cells. We can only speculate on the nature of this signal. The identification of clonal expansion of CD4+ T cells is a feature of atherosclerosis and type 1 diabetes; however, the identification of key antigens in these disease settings has also been elusive. Our studies suggest that therapeutic interventions that target APC-T cell communication may play a role in the treatment of insulin resistance and type 2 diabetes. In mice, MHC II anti-
body blockade decreased CD4+ATTs, but did not result in significant improvement in glucose tolerance (Morris et al., 2013). This may be due to improper dosing or window of intervention and will need to be revisited in light of our current work. Nonetheless, our findings suggest an overlap between efforts to block TCR signaling and costimulation in autoimmune diseases such as multiple sclerosis and type 1 diabetes and obesity-associated metainflammation (Podojil and Miller, 2009). It also implies that antidiabetic treatments and therapies targeting adipokines such as RBP4 may play critical roles in modulating APC function of resident and CD11c+ ATMs.
An important implication of our MMKO mouse model is that the ATM may be a unique cells that if targeted can provide beneficial effects in peripheral tissue responses without altering systemic T cells. Macrophages are thought to have less potent APC function than classical DCs. Our data suggest that adipose tissue may be a unique environment dominated by ATMs with strong APC activity that partner with T cells to establish an interaction network that perpetuates metainflammation.
EXPERIMENTAL PROCEDURES Animal Studies
C57BL/6J, LysMCre (B6.129P2-Lyz2tm1cre1'7J), MHC IIf/f (B6.129X1-H2-Ab1tm1Koni/J), CD11c-DTR (B6.FVB-Tg(Itgax-DTR/EGFP)57Lan/J), and OT-II (B6.Cg-Tg(TcraTcrb)425Cbn/J) mice were purchased from the Jackson Laboratory. MHC IIKO (B6.129-H2-Ab1tm1Gru N12) and WT (B6.SJL-Pfprca/ BoyAiTac) male mice were purchased from Taconic. CD11c-mCherry mice were kindly provided by Dr. Kamal Khanna (University of Connecticut). Macro-phage-specific MMKO mice were generated by breeding LysM-Cre with MHC IIf/f mice. Cre-negative MHC IIf/f littermates were used as control. Male mice were ad libitum fed a normal diet (4.5% fat; PMI Nutrition International) or fed a HFD consisting of 60% fat (Research Diets) beginning at 6 weeks of age. All mice procedures were approved by the University Committee on Use and Care of Animals at the University of Michigan and were conducted in compliance with the Institute of Laboratory Animal Research Guide for the Care and Use of Laboratory Animals.
Bone Marrow Transplantation
Bone marrow transplants were performed as described (Singer et al., 2013). Reconstitution was confirmed 6 weeks after transplantation, and recipient animals were fed either a ND or HFD for 8 weeks. For CD11c+ cell depletion, DT (Sigma-Aldrich) was administered IP (4 ng/g body weight) into chimeric CD11c-DTR mice every other day for 2 weeks while on diet. Control chimeric mice were injected IP with PBS.
Metabolic Evaluation
Body weights were measured weekly, and body composition was determined using a Minispec NMR analyzer (Bruker Optics). GTTs and ITTs were performed after a 6 hr fast. For GTTs, mice were injected IP with D glucose (0.7 g/kg). For ITTs, mice were injected IP with human insulin (1 U/kg). For both GTTs and ITTs, blood glucose concentrations (mg/dl) were measured 0, 15, 30, 45, 60, 90, and 120 min after injection. Serum insulin (Crystal Chem) and MCP-1 (R&D System) were measured by ELISA. Nonesterified fatty acids in serum samples were measured using serum/plasma-free fatty acid detection kits (Zen Bio). Total triglycerides were extracted from frozen liver samples (200 mg) and measured by colorimetric enzyme assay.
Confocal Microscopy
After consent, human omental adipose tissue samples were collected intrao-peratively from patients undergoing bariatric surgery at the University of Michigan. All human use protocols were approved by the University of Michigan Institutional Review Board. Immunofluorescence staining was performed as
described (Martinez-Santibafiez et al., 2014) using the following antibodies: anti-HLA-DR (clone LN3, eBioscience), anti-CD206 (clone 309210, R&D Systems), anti-Caveolin (clone 2297, BD), anti-CD4 (clone A161A1, Biolegend). Images were collected using an Olympus Fluoview 100 laser scanning confocal microscope.
Intravital Two-Photon Microscopy
The day before intravital microscopy, 2.5 x 106 CFSE-labeled OTII CD4+ T cells were adoptively transferred IP into recipient CD11c-mCherry mice. BSA or OVA (100 mg/mouse) was administrated 2 hr before imaging. Mice were anesthetized with pentobarbital (60 mg/kg body weight). Gonadal adipose tissue was surgically exposed and positioned in custom-built heated stage for intravital microscopy. Two-photon imaging was performed with Leica 5P MP confocal microscope (Leica Microsystems) equipped with 20x numerical aperture objective with excitation with a Mai-Tai Ti:Sapphire laser (Spectra Physics) tuned to 800 nm. Emitted fluorescence was collected using a two-channel nondescanned detector. For four-dimensional analysis of cell trafficking, stacks of 20 section (z step = 1 mm) were acquired every 1 min to provide an imaging volume of up to 20 mm in depth. Sequences of image stacks were transformed into volume-rendered four-dimensional movies using Imaris software (Bitplane), and the spot analysis was used for semiauto-mated tracking of cell motility in three dimensions by using the parameters of autoregressive motion as an algorithm 8 mm spot diameter and 20 mm maximum distance. Imaris software (Bitplane) was used to calculate T cell velocity, T cell displacement, and CD11c cell-CD4 cells interactions.
Isolation of Adipose Tissue SVCs and Flow Cytometry Analysis
SVCs from adipose tissues were isolated as previously described (Cho et al., 2014).
Cells were incubated in Fc Block for 10 min on ice and stained with indicated antibodies for 30 min at 4°C. Stained cells were washed twice in PBS and fixed in 0.1% paraformaldehyde before analysis. For intracellular staining and bromodeoxyuridine (BrDU), cells were stained as described before (Morris et al., 2013). Analysis performed on FACSCanto II Flow Cytometer or FACSAria III (BD Bioscciences) and analyzed with FlowJo software (Treestar).
Cell Culture
Bone marrow cells were isolated from mice by flushing of tibia and fibula and differentiated into bone marrow-derived macrophages (BMDMs) as described (Singer et al., 2013). Differentiation was confirmed by demonstrating F4/80 expression by flow cytometry. Cells were placed in 10% serum media for 24 hr prior to treatment with LPS (10 ng/ml) for 6 hr.
Gene-Expression Analysis
RNAfrom tissues and cells was extracted using RNeasy Midi Kits (QIAGEN), and cDNA was generated from 0.5-1.0 mg total RNA using high-capacity cDNA reverse transcription kits (Applied Biosystems). Power SYBR Green PCR Master Mix (Applied Biosystems) and the StepOnePlus System (Applied Biosystems) were used for real-time quantitative PCR. Arbp expression was used as an internal control for data normalization. Samples were assayed in duplicate, and relative expression was determined using the 2~iiCT method.
Immunoblotting Analyses
Mice were fasted for 6 hr and stimulated with insulin (3 U/kg body weight) or PBS IP. Liver and eWAT extracts were prepared 5 min after stimulation by homogenization in lysis buffer containing 50 mM Tris (pH 7.5), 1% Nonidet P-40, 150 mM NaCl, 2 mM EGTA, 1 mM NasVO*,, 100 mM NaF, 10 mM Na4P2O7, and protease inhibitors. Proteins was separated by SDS-PAGE, immunoblotted with indicated antibodies, and visualized using the Odyssey infrared imaging system (Li-Cor Bioscience). Phospho-AKT (Ser473) and AKT antibody were purchased from Cell Signaling Technology.
Statistical Analysis
All values are reported as mean ± SEM. Differences between groups were determined using unpaired, two-tailed Student's t test or one-way ANOVA with Tukey post hoc tests with GraphPad Prism 5 software. p values less than 0.05 were considered significant.
SUPPLEMENTAL INFORMATION
Supplemental Information includes six figures, two tables, and two movies and can be found with this article online at http://dx.doi.org/10.1016/j. celrep.2014.09.004.
AUTHOR CONTRIBUTIONS
K.W.C, D.L.M., J.L.B., K.S., L.G., B.Z., G.M-S., and C.N.L. performed, designed, and interpreted the experiments performed. K.A.M. and R.W.O. contributed to human sample collection and analysis. K.W.C. and C.N.L prepared the manuscript. All authors approved the final manuscript.
ACKNOWLEDGMENTS
This work was carried out with support from the NIH/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK; DK090262 and DK092873 to C.N.L., DK095050 and DK097449 to R.W.O.) and the American Diabetes Association (07-12-CD-08). Trainees were supported by NIH National Institute of Allergy and Infectious Diseases Experimental Training in Immunology T32 AI007413-19 (B.Z), NIH NIDDK F32 DK091976 (D.L.M.), and an NIH Minority Training Supplement (DK090262-S1 to G.M-S.). This work utilized Core Services from the Michigan Nutrition and Obesity Research Center supported by grant DK089503 of NIH to the University of Michigan. This work utilized the Microscopy Core of the Michigan Diabetes Research Center funded by NIH 2P30-DK20572 from the NIDDK.
Received: April 7, 2014 Revised: July 18, 2014 Accepted: August 28, 2014 Published: October 9, 2014
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