Mol Cell Toxicol (2010) 6:143-149 DOI 10.1007/s13273-010-0021-6
ORIGINAL PAPER
Systemic immunity of obese-diabetes model (db/db) mice Sang-eun Lee1, Ik-soon Jang3, Jun-soo Park4, Ji-Hae Lee2, Seung-Yeul Lee2, So-young Baek1, Seung-hoon Lee2,* & Hyunah Lee1,*
Received: 18 December 2009 / Accepted: 14 April 2010 �The Korean Society of Toxicogenomics and Toxicoporeteomics and Springer 2010
Abstract Obesity has recently been defined as a chronic inflammatory disease and is considered as a major cause of adult health problems including incurable diseases like diabetes and cancer. In this study, the systemic immunity, including the innate and adaptive immunity parameters, of naturally occurring (leptin receptor mutation) obesity-diabetes mice (db/db) was examined to increase our knowledge of these mice for obesity-related studies. Severe fatty liver with blood engorgement was observed in the db/db mice. Compared to background C57BL/6J mice, the adaptive immunity, as measured by mitogen-induced T and B cell proliferation and cytokine release, was significantly suppressed in db/db mice. However, significant upregulation of innate immune-inflammatory parameters including macrophage function and NK cell activity was observed in db/db mice without external stimulation. These data conclusively confirm the systemic immune-inflammatory micro-environment with suppressed adaptive immunity of db/db mice, which may cause the secondary obesity-related life-threatening diseases like diabetes or cancer. Keywords Obese-diabetes model, Systemic immunity, db/db mice
1
Clinical Research Institute, Samsung Medical Center Sungkyunkwan University School of Medicine, 50 IL-Won Dong, Gang-Nam Gu, Seoul 135-710, Korea 2 Department of Life Science, Yong-In University, 470 Samga Dong, Cheoin Gu, YongIn 449-714, Korea 3 Korea Basic Science Institute, Daejeon 305-333, Korea 4 Division of Biological Sciences and Technology, Yonsei University, Wonju 220-701, Korea *These authors contributed equally to this work Correspondence and requests for materials should be addressed to S. H. Lee (
[email protected]) and H. Lee (
[email protected])
In modern society, obesity is considered a disease that causes incurable adult health problems including type II diabetes, cardiovascular disorders and cancer1-5. Recent findings indicate that obesity is a chronic inflammatory disease3,6,7. Increased NF-κB nuclear binding is known to be related with inflammation in obesity3. Inflammation-related molecules like adiponectin, cytokines (TNF-α, IL-1β or IL-6) and chemokines are secreted from white adipocytes and macrophages. The infiltration of macrophages into the adipose tissue observed in the obese state worsens the inflammatory situation of white fat3. Plasma-circulating adipokines and pro-inflammatory cytokines that are secreted from the cells with monocyte/macrophage phenotype may represent a mechanism for several obese-related adult health problems like type II diabetes3-5,9. Along with a clear association between immune-inflammatory responses and obesity, systemic cellular immune modulatory responses other than inflammation in obese targets have been also reported. Obesity-related systemic immune responses such as cytokine dysregulation and lipotoxicity can cause insulin resistance and metabolic disease, which increase the lethality in patients through internal organ destruction or infection 9,10. Evidences from human as well as animal model suggest that obesity may be associated with immunodeficiency state including significantly impaired T cell responses5-8. Interestingly, modulation of regulatory T cell responses confirmed by increased transcription factor FoxP3 in the lymphocytes was observed in leptin-deficient obese (ob/ob) mice2. In this study, the systemic immunity, including the innate and adaptive immune parameters, of naturally occurring obese-diabetic mice (db/db; leptin receptor mutation) was examined to increase our basic knowledge for obesity-related studies. Leptin is adipocyte-secreted hormone that controls the weight centrally. The leptin receptor is
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expressed on the central nervous system as well as on the hematopoietic and peripheral immune system11. Leptin/leptin receptor signal pathway is known to be critical to modulate the lymphocyte proliferation, apoptosis and cytokine secretion12,13. Thus, to resolve the health problems related to obesity, research using leptin (receptor) deficient animal model may proper by expand our understanding of obesity and related diseases in terms of the induction mechanism or development of therapeutics. Histopathological changes of internal organs
At the beginning of the experiment, the average body weight of db/db mice was about twice that of normal
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Figure 1. Body weight of C57BL/6J & db/db mouse was compared. Body weight was expressed as mean±se of 10 and 14 mice for each wild and db/db mice group. Asterisk means statistical difference between two groups with P‹0.05.
A
C57BL/6J mice (Figure 1). Fatty liver with necrosis of hepatic tissue was discovered in obese mice. Histopathological changes of several organs including liver were confirmed by H&E staining. In microscopic observation, fatty liver and blood engorgement were observed in db/db mice (Figure 2). No significant changes were detected in the kidney and lung (data not shown). In general, severe internal organ atrophy was observed with packed viceral fat in db/db mice. Changes in immune-inflammatory (innate immunity) parameters
Obesity is defined as chronic inflammatory disease in which functional and proportional changes of macrophage (MP) or natural killer (NK) cells are deeply involved. We confirmed this fact by observing the significantly increased NK cell and MP activities in db/db mice compared to those in background C57BL/ 6J mice (Figure 3A & B). Without exposure to external stimuli, splenic NK cell activity was about 10 times higher in db/db mice than in C57BL/6J background mice (Figure 3A). The proportion of phagocytic macrophages (F4/80+ cells engulfing dextran-FITC) also significantly higher in db/db mice without stimuli (F4/80+dextran-FITC+ cells: 5.08% vs. 0.76% for db/db vs. C57BL/6 mice, respectively) (Figure 3B). Such responses were not observed from bone marrow progenitor cells in db/db mice. Adaptive immunity in db/db mice
In order to compare the immune cell functions of normal C57BL/6J mice and db/db mice, mitogen-induced T & B cell proliferation was observed. In db/db mice, splenic lymphocyte proliferation was significantly
B
Figure 2. H&E staining of liver tissue from the (A) C57BL/6J & (B) db/db mouse: severe fattyliver was observed in db/db mice. Stained tissue was observed and photographed by LEICA DM3000 by ×200 magnification.
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Figure 3. (A) Splenic NK cell activity in C57BL/6J (wild type) and db/db mice were measured as was described in “Materials and Methods”. Results are expressed as mean % of NK activity of triplicate samples: bars indicate standard error. (B) Macrophage phagocytic activity was measured by flow cytometry. Splenocytes and bone marrow hematopoietic progenitor cells were concomitantly labeled with F4/80-PE and dextranFITC. Percentage of PE & FITC double positive cells was determined as the phagocytic macrophages.
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Figure 4. Mitogen-induced lymphocyte proliferation was measured. ConA, a T cell mitogen and LPS, a B cell mitogen was co-cultured with either splenic lymphocytes or bone marrow hematopoietic progenitor cells to analyze the lymphocyte functions. 3H-thymidine incorporation assay was used to define the proliferative ability of cells. The statistical significance between C57BL/6J (wild type) and db/db mice group (P‹0.05) was represented by asterisk.
defective compared to that in C57BL/6J mice (Figure 4A). Baseline level of splenic lymphocyte proliferation in db/db mice was 1/5 of that in C57BL/6J mice. Stimulation with ConA, a T cell mitogen, or with LPS, a B cell mitogen, did not induce lymphocyte proliferation from the db/db mouse spleen (In C57BL/6J mouse spleen vs. db/db mouse spleen: 350% vs. 42% by ConA, 194% vs. 39% by LPS, respectively). Unlike the responses observed with splenic lymphocytes, LPSinduced proliferation was significantly higher in bone
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marrow progenitor cells from db/db mice (199% vs. 315 % for C57BL/6J mice vs. db/db mice, respectively) (Figure 4B). Phenotype analysis of splenic lymphocytes
Functional change of immune cells in db/db mouse spleen was correlated with the proportional alteration in immune cell populations, which were measured by flow cytometric analysis. Proportion of innate immune cells including macrophage CD11b+Macs3+; 4.6% vs. 15.1% for C57BL/6J vs. db/db mice, respectively), dendritic cells (CD11c+) and NK cell (DX5 +) was higher in db/db mouse spleen without external stimula(A)
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tion (Figure 5A). On the other hand, the proportion of adaptive immunity cells, such as T cells (CD3+; 41.1% vs. 18.0% for C57BL/6J vs. db/db mice, respectively) and B cells (CD19+), was significantly lower in db/db mouse spleen. Interestingly, T cell subsets, including helper T (CD4+; 27.5% vs. 11.2% for C57BL/6J vs. db/db mice, respectively) and cytotoxic T (CD8+; 14.3% vs. 8.2% for C57BL/6J vs. db/db mice, respectively) cells, were also significantly decreased in the obese model (Figure 5B). However, the proportion of immune regulatory cells, including myeloid-derived suppressor cells (MDSC: Gr-1 +CD115 +; 1.7% vs. 10.1% for C57BL/6J vs. db/db strain, respectively)
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Figure 5. Surface phenotype expression was measured in splenic lymphocytes and bone marrow hematopoietic progenitor cells. Cells were harvested and double stained with FITC/PE conjugated each surface marker antibodies; (A) anti-mouse DX5/CD11c (NK/dendritic cells), CD11b/Mac-3 (resting/activated macrophage), (B) CD8a/CD4 (T cell subset), CD19/CD3 (B & T cell), (C) Gr-1/CD115 (myeloid suppressor cells), CD25/CD4 (regulatory T cell).
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Figure 6. Cytokine secretion into the plasma of C57BL/6J and db/db mice was measured. Mouse plasma samples were obtained by venous puncture. Level of cytokines was measured by multiplex cytokine analysis kit (Upstate Cell Signaling Solutions, USA) and analyzed using the MasterPlex QT 1.0 system. Statistical significance between two groups was expressed as asterisk meaning P‹0.05.
and naturally occurring regulatory T cells (Treg: CD4+ CD25+; 4.3% vs. 10.3% for C57BL/6J vs. db/db strain, respectively), was much higher in db/db mouse spleen (Figure 5C). MDSC and Tregs are known to suppress the function of effector cells including T cells. Data suggest that the baseline systemic immunity of db/db mice was significantly suppressed in the adaptive arm but inflated in the innate (inflammatory) arm. Cytokine secretion into the blood
Levels of cytokines secreted into the blood were compared between db/db and background C57BL/6 mice. The levels of T cell-produced cytokines such as IL-2, IL-10 and IL-12, a T cell stimulating factor produced by dendritic cells or macrophages to induce antigenspecific cellular immunity were significantly lower in db/db mice without external stimulation (C57BL/6J vs. db/db mice: IL-2: 18 pg/mL vs. 1 pg/mL, IL-10: 67 pg/mL vs. 22 pg/mL, IL-12: 213 pg/mL vs. 48 pg/mL) (Figure 6). This phenomenon is well correlated with suppressed T cell proliferative function and the lower splenic proportion of T cells in db/db mice. But, interestingly, immune-inflammatory cytokines including TNF-α and INF-γ secreted into the plasma were below the level of detection (Figure 6).
Discussion Obesity is considered a chronic immune-inflammatory disease that causes incurable adult health problems
such as type II diabetes, cardiovascular disorders and cancer1-5. Several animal models have been used to study obesity and related disorders; these include mice with leptin (ob/ob) or leptin receptor (db/db) mutation. However, data regarding altered baseline immunity in obesity model mice, which may provide basic knowledge to verify the obesity-related studies, has not been found. In this study, the baseline systemic immuneinflammatory state without external stimuli was investigated in db/db mice, a strain with a spontaneous mutation of leptin receptor that results in obesity and diabetes. Growing db/db mice developed obesity as determined by increasing body weight and accumulation of body fat (Figure 1). Significant fatty liver with blood engorgement was observed in db/db mice but not in the livers of control mice, C57BL/6J background strain (Figure 2). Compared to those in control background mice, both systemic innate and adaptive immunity were significantly altered in db/db mice. In particular, macrophages, immune-inflammatory cells and natural killer cells in innate immunity were significantly activated in db/db mice without any external stimuli (Figure 3). The phenomenon was comparable to infected immunity in normal mice, which explains the abnormal immune-inflammatory state of the db/db mice. In contrast to the innate immunity, the adaptive immune parameters were significantly suppressed in db/db mice. The functional quality and the quantity of splenic lymphocytes in db/db mice were 1/5 that in C57BL/6J mice and were accompanied by significantly lower blood cytokine levels. The proliferative function as well as the mitogen-sensitivity of splenic lymphocytes was severely impaired in db/db mice (Figure 4). Among T cells, although the effector T cell function and number were decreased in the spleen, the naturally occurring regulatory T cell proportion was increased in the db/db mice. Also the proportion of Gr-1 +CD115 + MDSC, a bone-marrow derived immune suppressor, was significantly increased in the db/db mouse spleen. Increased immune suppressive cell proportions in the db/db mouse spleen may be one of the mechanisms explaining inhibited cellular immunity in this animal (Figure 5). Data from our study confirmed that the immune micro-environment of obese mice is in an inflammatory state3,6,7 and that their adaptive cellular immunity is significantly suppressed5-8. This phenomenon may suggest decreased sensitivity of first-line defense mechanism to external stimuli in obese mice and, thus, impaired adaptive immunity to control the outside attack. The immune microenvironment of obese mice is therefore fragile for induction of secondary diseases like diabetes or cancer. Data from this study regarding
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the systemic immunity of the obese-diabetes mouse model (db/db) may expand our understanding of obesity and related diseases in terms of the induction mechanism or development of therapeutics.
Materials & Methods Animal
Specific pathogen-free female db/db mice as an obese model animal and C57BL/6J mice, a background control mouse of 4-5 weeks old have been obtained from Charles River Japan, Inc. (Yokohama, Kanagawa, JAPAN). db/db mice delivered by spontaneous mutation of leptin receptor (a/a + Lepr db/ + Lepr db) and without diet control, became obese around 3-4 weeks old. Elevation of plasma insulin level begins at 10-14 days old and of plasma sugar at 4-8 weeks old. Fivesix weeks old mice were used for this experiment. Specific pathogen-free C57BL/6J mice and db/db mice were provided with water and food, ad libitum and quarantined under 12 h light : 12 h dark photoperiod in the animal care facility and animal care was performed following the ILAR guideline. The mice were acclimated for at least one week before any experiments were conducted. Reagents
RPMI-1640 medium, fetal bovine serum and penicillinstreptomycin were obtained from GIBCO laboratories (Grand Island, NY); ConA (Concanavalin A, from Canavalia ensiformis) and LPS (Lipopolysaccharides, from Escherichia coli 055:B5), Dextran-FITC were obtained from SIGMA Chemical Co. (St.Louis, MO, USA). [3H]-Thymidine and Na251CrO4 were purchased from Perkin Elmer Corp. (Norwalk, CT, USA). Antibodies for flow cytometric phenotyping were obtained from eBioscience (Sandiego, CA, USA). Cell line
Target cell of natural killer (NK) cell activity, YAC-1 mouse lymphoma cell line was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cell line was maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (complete medium). Immune cell proliferation assay
To define the function of immune cells from splenic lymphocytes or hematopoietic progenitor cells from bone marrow, induction of proliferation by mitogens
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like ConA (for T cells) or LPS (for B cells) was observed by 3H-Thymidine incorporation assay. Responder splenic lymphocytes (1×106 cells/mL) from db/db mice were incubated with 1 μg/mL final concentration of either ConA or LPS for 96 hrs. Eighteen hours before harvest, 1 μCi 3H-Thymidine was pulsed. Cells were harvested on the glass fiber filter (Whatman, Maidstone, England) using PhD. Cell harvester (Cambridge Technology, Inc., Cambridge, MA, USA). Filters were covered with scintillation cocktail (Beckman, Fullerton, CA, USA) and the radioactivity incorporated into the proliferating cells was measured by β-scintillation counter (Beckman LS6500, Fullerton, CA, USA). Phenotype analysis by flow cytometry
Surface markers of immune cells from splenic lymphocytes or hematopoietic progenitor cells from bone marrow were determined. Single cells from the db/db mouse splenocytes or bone marrow progenitors (1× 105 cells/100 μL) were incubated with fluorescence (FITC or PE) labeled surface antibodies in PBS with 0.1% sodium azide and 1% FBS (PBS-CS) for 40 min. at 4� C. Either FITC or PE-conjugated anti-mouse antiCD19 (for B cells), CD3, CD4, CD8, CD25 (for T cell subsets), Mac1, Mac3, F4/80 (for macrophages), DX-5 (NK cells), CD11c (dendritic cells) was selected to analyze the alterations. Within 2 hr after the antibody labeling, cells in the 500 μL PBS-CS were analyzed in the flow cytometer (FACSVantage, Becton-Dickson, Mountain View, CA). NK activity
NK activity was measured by 51Cr-release assay from the Na251CrO4 labeled YAC-1 (1×104/100 μL) target cells (T) incubated with effector splenic lymphcytes (E) for 4 hrs. Radioactivity in the supernatants released from the target cells was measured by Wallac 1470 Wizard gamma counter (Finland). Spontaneous release (SR) and total release (TR) were measured in the supernatants of target cells incubated with either culture medium or 1 N HCl. Percent NK-cell activity was calculated as follow. % NK cell activity Experimental cpm-SR cpm =mmmmmmmmmmmmmmmmmmmmmmmmmmmm×100 TR cpm-SR cpm Macrophage phagocytosis
Phagocytic ability of F4/80+ macrophages was determined by measuring Dextran-FITC uptake level with flow cytometer. Splenic lymphocytes were labeled with both Dextran-FITC and anti-F4/80 (PE) antibodies and analyze the double positive (F4/80+ and Dex-
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tran + ) cells with flow cytometer (FACS Vantage, Becton-dickenson, Mountain View, CA) as phagocytic macrophages. Cytokine microbead assay
Cytokines secreted into the blood were measured by microbead assay using plasma as a sample. Mouse blood was obtained by ocular-venous puncture using non-heparinized capillary tube and immediately centrifuged at 3,000 rpm for 10 min to separate the plasma, aliquoted, and stored at -70� C until assayed. Multiple cytokine analysis kit was obtained from Upstate Cell Signaling Solutions (Lake Placid, NY). Millipore multi-screen 96 well filter plates (Bedford, MA) were used for multiplex cytokine kits. Assays were run in triplicate according to the manufacturers’ protocol. Data was collected using the Luminex-100 system Version 1.7 (Luminex, Austin, TX). Data analysis was performed using the MasterPlex QT 1.0 system (MiraiBio, Alameda, CA). Statistical analysis
Each experimental group contained 6-7 mice for each experiment. The experiments were repeated 2-3 times by the same protocol. Data expressed as mean± standard error (SE). Significant differences among the treatment groups were calculated by Student’s t-test. Differences were considered significant at P⁄0.05. Acknowledgements This study has been supported by the 2008 grant from Korean Food and Drug Administration (#08152 toxicity control 423).
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