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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Objective

To determine whether the expression of key asthma related genes, IL-4, LIGHT, LTBR, MMP-9, CCR-2, and ADAM-33 in mononuclear cells and the plasma concentration of nitric oxide metabolites (NOM) and MMP-9 are increased in the obese, obese type 2 diabetics (T2DM) and in morbidly obese patients prior to and after gastric bypass surgery (RYGB).

Design and Methods

The expression of these genes in MNC and plasma concentrations of these indices was measured in healthy lean and in obese with and without T2DM and following RYGB in obese T2DM.

Results

The expression of IL-4, MMP-9, LIGHT and CCR-2 and plasma NOM concentrations was significantly higher in the obese subjects and in obese T2DM patients than in normal subjects. The expression of IL-4, LIGHT, MMP-9, and CCR-2 expression was related to BMI and HOMA-IR. The expression of IL-4, LIGHT, LTBR, ADAM-33, MMP-9, and CCR-2 fell after RYGB surgery as did plasma concentrations of MMP-9 and NOM.

Conclusions

Obesity with and without T2DM is associated with an increase in the expression of IL-4, LIGHT, MMP-9 and CCR-2; plasma NOM and MMP-9 concentrations are also increased. Following RYGB surgery and weight loss, the expression of these factors in MNC and plasma concentrations falls significantly.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The risk of asthma in the obese is two to three times greater than that in normal subjects ([1]). A recent report has shown that there is an increase in the prevalence of asthma in patients with type 2 diabetes (T2DM) by over 200% but when corrected for BMI, the increase though significant, was by only 8% ([2]). Thus obesity appears to be the major contributor to the pathogenesis of asthma. The increase in abdominal adiposity may affect lung volumes and restrict pulmonary and bronchial expansion during inhalation ([3]). In addition, since obesity is characterized by chronic inflammation and oxidative stress, with an increase in the mediators of innate immunity ([4-7]), we asked whether obesity is also associated with an increase in the expression of Th2 cytokines like IL-4 which characterize allergic inflammation ([8-10]). An increase in the expression of IL-4 is known to occur in bronchial asthma. IL-4 induces an increase in IgE and mediates other important changes which contribute to the pathogenesis of asthmatic inflammation. Soluble IL-4 receptor has been infused in asthmatic patients to successfully reverse asthmatic inflammation by diverting IL-4 away from its cellular receptors ([11, 12]). Thus, a specific interference with the biological action of IL-4 results in the suppression of the inflammatory and clinical manifestations of asthma.

T2DM is also characterized by chronic inflammation and insulin resistance which may be attributable to interference in insulin signal transduction caused by inflammatory mediators ([13]). A large study has recently shown that the risk of asthma in patients with T2D is two times greater than that in normal subjects ([14]). It is therefore possible that T2DM may also be associated with an increase in the expression of IL-4.

Other inflammatory mediators which play a role in asthma include matrix metallo-proteinases (MMP), including MMP-9 and ADAM-33 (A Disintegrin and Metalloproteinase-33); and chemokines including eotaxin and MCP-1 ([15-17]). In addition to the possible contribution of these pro-inflammatory mediators, there is an increase in nitric oxide (NO) and isoprostane content in exhaled air in asthmatics when compared to normal subjects ([18]). The increase in NO is likely due to the activation of iNOS in bronchial macrophages and is thought to relate to the clinical activity of asthma ([19]). Increased isoprostane generation is a reflection of increased oxidative stress which also characterizes asthmatic inflammation ([20]). More recently, two other pro-inflammatory genes have been shown to be associated with asthma. They are lymphotoxin-β receptor (LTBR) and its ligand, LIGHT ([21]). They participate in the crucial process of bronchial remodeling in chronic asthma as do matrix metalloproteinases like MMP-9. LIGHT mediates bronchial wall fibrosis and smooth muscle hyperplasia while also increasing bronchial hyperresponsiveness ([21]). Its profibrotic actions are mediated through TGFβ and IL-13.

Our recent work shows that in morbidly obese patients, gastric bypass surgery and weight loss are associated with a reversal of oxidative stress and inflammation ([22]). It is, therefore, possible that there may be a reduction in the expression of asthma related genes as a part of this overall effect.

Based on the above points, we hypothesized that ([1]) the expression of IL-4, LTBR, LIGHT and ADAM-33, and the plasma concentration of NOM are increased in obesity and in T2DM; and ([2]) the expression of the above asthma related genes diminishes significantly after Roux-en-Y gastric bypass (RYGB) surgery.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Subjects

Lean subjects and patients with obesity and T2DM

Three groups (Table 1) of normal weight controls (n = 22, body mass index (BMI) = 23.1 ± 0.6 kg m−2), obese patients with and without diabetes (n = 23, BMI = 35.5 ± 1.8 kg m−2) and morbidly obese patients with T2DM (n = 15; mean BMI: 52.1 + 13.0 kg m−2) were included in the study. A subgroup of the obese subjects had type 2 diabetes (T2DM) (n = 11, BMI = 35.7 ± 1.8 kg m−2) and data were stratified accordingly. Baseline characteristics for these patients are presented in Table 1. Patients with T2DM were not on insulin or thiazolidinediones. Most of the diabetic patients (8 out of 11) but not the nondiabetic obese were on a stable dose of statins. None of the normal subjects or patients was on NSAIDs or antioxidants. None of the patients had a known history of asthma or cardiovascular disease. None of the patients or normal subjects was a smoker. The protocol was approved by the Human Research Committee of the State University of New York at Buffalo. An informed consent was signed by all subjects. Fasting blood samples were collected for baseline comparison between the groups. The morbidly obese T2DM patients underwent RYGB and were followed up for 6 months following the surgery. The morbidly obese patients were required to have a minimum of 3 months of stable ACEI/ARB, statin, and T2DM therapy, defined as no greater than a one-step dose increase or decrease (i.e., metformin from 1000 to 500 mg or glyburide 10 to 5 mg). Insulin requirements were not changed by >25%. Subjects were excluded if they required chronic aspirin, NSAID's or systemic corticosteroids. Fasting blood samples were collected before and 6 months following the RYGB procedure. The study was approved by the Institutional Review Board of the Catholic Health System. Each participant signed an informed consent. (ClinicalTrials.gov number, NCT00960765).

Table 1. Demographic data at baseline for comparison between lean and obese subjects (n = 22 and 38, respectively) and in subgroups of obese group with and without T2DM and at 6 months following RYGB surgery in the morbid obese T2DM subgroup
     Morbid Obese/T2DMMorbid Obese/T2DM
 LeanAll obeseObese Non T2DMObese/T2DMBefore RYGB6 months following RYGB
  1. ANOVA was followed by Dunn's pairwise test: *=P < 0.05, (compared to lean group) and # =P < 0.05 (obese/T2DM compared to obese non T2DM);  = P < 0.05 compared to morbidly obese/T2DM baseline by paired t test. Values are reported as mean ± SE. NA = not available.

N223812111515
Age (yrs)39 ± 543 ± 742 ± 641 ± 445 ± 9 
Gender (m/f)9/1315/235/75/65/10 
HbA1cNA6.7 ± 0.75.4 ± 0.37.0 ± 0.5#7.9 ± 1.4#6.3 ± 0.8
BMI (kg m−2)23.1 ± 0.642.3 ± 3.0*35.2 ± 1.8*35.7 ± 1.8*52.1 ± 4.5*#40.4 ± 3.6
Weight (kg)68 ± 7123 ± 17*110 ± 9*108 ± 9*150 ± 33*#115 ± 28
Glucose (mg dL−1)75 ± 4113 ± 7*80 ± 4113 ± 9*#148 ± 8*#101 ± 4
Insulin (μIU mL−1)4.5 ± 0.617.8 ± 4.1*14.2 ± 4.3*17.7 ± 4.9*18.5 ± 2.2*8.6 ± 1.0
HOMA-IR0.85 ± 0.186.0 ± 1.5*3.2 ± 1.4*6.1 ± 2.0*#7.9 ± 1.4*#2.1 ± 0.3
Triglycerides (mg dL−1)95 ± 12179 ± 31*157 ± 12*166 ± 13*210 ± 53*132 ± 20
MNC isolation

Blood samples were collected in Na-EDTA and carefully layered on Lympholyte medium (Cedarlane Laboratories, Hornby, ON). Samples were centrifuged and two bands separate out at the top of the RBC pellet. The MNC band was harvested and washed twice with Hank's balanced salt solution (HBSS). This method provides yields >95% MNC preparation.

Quantification of mRNA in MNC by RT-PCR

Total RNA was isolated using commercially available RNAqueous®-4PCR Kit (Ambion, Austin, TX). Real Time RT-PCR was performed using Stratagene Mx3000P QPCR System (La Jolla, CA), Sybergreen Master mix (Qiagen, CA) and gene specific primers for IL-4, MMP-9, MCP-1, LIGHT, LTBR, LTB, ADAM-33, and CCR-2 (Life Technologies, MD). All values were normalized to the expression of a group of housekeeping genes including actin, ubiquitin C and cyclophilin A. The intra- and interassay coefficient of variation (CV) for IL-4 expression by PCR were 6 and 10%, respectively. CV for the other RT-PCR assays ranged from 5 to 12% (intra-assay) and 7 to 13% (inter-assay).

Plasma measurements

Glucose concentrations were measured in plasma by YSI 2300 STAT Plus glucose analyzer (Yellow Springs, OH). ELISA was used to measure insulin (Diagnostic Systems Laboratories, Webster, TX), MMP-9 and LIGHT (R&D Systems, MN). Plasma concentrations of nitric oxide metabolites (NOM: NO2/NO3) were measured by Griess reaction using a colorimetric assay kit from R&D systems (Minneapolis, MN). The CVs for these assays ranged from 2 to 5% and 4 to 8% for intra- and interassay variations, respectively.

Statistical analysis

Statistical analysis was conducted using SigmaStat 11 software (SPSS, Chicago, IL). All data are represented as mean ± SE. Demographic variables and baseline levels of inflammatory mediators in lean, obese and obese T2DM were compared using ANOVA followed by Dunn's test for pairwise comparisons. Changes from baseline in the RYGB group were compared with paired t test. Pearson's product-moment correlation coefficient was calculated to study dependency between variables. P < 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Expression of asthma related pro inflammatory mediators in MNC of obese and T2DM

The mRNA expression of IL-4, MMP-9, and CCR-2 was significantly higher in MNC from all obese subjects by 112, 57, and 134%, respectively, (P < 0.05, ANOVA on Ranks with Dunn's post hoc comparisons) above baseline when compared to normal subjects. The expression of IL-4, MMP-9 and CCR-2 was the highest (by 143, 77, and 145%, respectively) in the morbidly obese diabetic subgroup compared to lean subjects. IL-4 expression in the morbidly obese T2DM was significantly greater than that in obese non T2DM subgroup (Figure 1A). The expression of LIGHT in the MNC from all obese subjects was significantly greater (by 39%) than that in lean controls. LIGHT expression was higher in obese with T2DM and in the morbidly obese with T2DM subgroups when compared to lean subjects. The group with the highest expression of LIGHT was the morbidly obese group with T2DM (by 58%) (Figure 1B). The expression of LIGHT was trended to be higher than the controls in obese group without diabetes but the difference was not significant. The expressions of LTBR and ADAM-33 in MNC were not significantly different between obese and control groups.

image

Figure 1. Relative basal mRNA expression of (A) IL-4, MMP-9 and CCR-2 and (B) LIGHT, LTBR and ADAM33 in MNC from lean and obese subjects (n = 22 and 44, respectively) and in subgroups of obese group with and without T2DM. ANOVA was followed by Dunn's pairwise test: *= P < 0.05, (compared to lean group); # =P < 0.05 (morbid obese/T2DM compared to obese non T2DM).

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Concentrations of asthma related mediators in obese and T2DM

Plasma concentrations of NOM and MMP-9 were significantly higher in the obese (by 34 and 66%, respectively, P < 0.05, ANOVA on Ranks with Dunn's post hoc comparisons) above baseline when compared to normal subjects (Figure 2A-B). NOM and MMP-9 concentrations were higher in all obese subgroups compared to lean subjects, with the highest levels in the morbid obese T2DM patients (by 44 and 104%, respectively). Plasma MMP-9 concentrations were significantly higher in the morbidly obese T2DM patients compared to other obese subgroups (Figure 2B). LIGHT concentration was significantly higher in the entire obese group by 70% when compared to normal subjects (Figure 2C). This was attributable largely to the markedly elevated levels in the morbidly obese since LIGHT concentration was not significantly different in the other obese groups when compared to controls. IL-4 was not detected in plasma, using commercially available kits, in any of the groups.

image

Figure 2. Plasma NOM (NO2/NO3) (A), MMP-9 (B), and serum LIGHT (C) concentrations in lean and obese subjects (n = 22 and 47, respectively) and in subgroups of obese group with and without T2DM. Values are reported as mean ± SE. ANOVA was followed by Dunn's pairwise test: *=P < 0.05, (compared to lean group); and #= P < 0.05 (morbid obese/T2DM compared to obese non T2DM),  = P < 0.05 (morbid obese/T2DM compared to obese T2DM).

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Relation of asthma-related mediators with BMI and HOMA-IR

BMI and HOMA-IR were related significantly to IL-4 mRNA expression, MMP-9 and LIGHT plasma concentrations (Figure 3). Additionally, BMI was related to LIGHT and CCR-2 mRNA expression in MNC (Figure 3).

image

Figure 3. Spearman Rank order correlation between BMI and relative mRNA expression of (A) IL-4, (B) LIGHT, and (C) CCR-2 in MNC, and with (D) plasma MMP-9 and (E) LIGHT concentrations and between HOMA-IR and (F) IL-4 mRNA, (G) serum LIGHT and (H) plasma MMP-9 concentrations from lean and obese subjects. mRNA expression is presented in Arbitrary Units. Regression coefficient (r) and statistical significance (P) are reported for each test.

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Effect of Roux-en-Y gastric bypass surgery on asthma related gene expression

Following RYGB there was a significant reduction in body weight, BMI, glucose, insulin, triglycerides, HbA1c, and HOMA-IR (Table 1) and in major oxidative and inflammatory mediators including NFκB and MNC ROS generation ([22]). In addition, RYGB caused a significant reduction in the mRNA expression in MNC of the asthma related mediators including IL4, ADAM33, LIGHT, and LTBR by 49% ± 6%, 20% ± 9%, 29% ± 8%, and 33% ± 7%, respectively, (P < 0.05) at 6 months following the surgery (Figure 4A). The surgery also induced a significant reduction in MMP-9, MCP-1 and CCR-2 mRNA expression in MNC by 59% ± 8%, 23% ± 6%, and 27% ± 8%, respectively, (Figure 4B, P < 0.05).

image

Figure 4. Percent change in (A) IL-4,LIGHT, LTBR, and ADAM33, and (B) MMP-9, MCP-1 and CCR-2 in mRNA expression in MNC from morbid obese T2DM patients before and 6 months following RYGB (N = 12). Data are presented as mean ± SE. *P < 0.05 by paired t test.

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Effect of Roux-en-Y gastric bypass surgery on asthma related circulating mediators

Following RYGB, there was a reduction in NOM (NO2/NO3) concentrations by 22% ± 9% (from 25.6 ± 2.1 to 20.87 ± 1.9 μM, P < 0.05, Figure 5) and in plasma MMP-9 concentrations by 26% ± 6% (from 79.2 ± 5.4 to 58.7 ± 6.4 ng ml−1, P < 0.05, Figure 5). LIGHT concentration, however, did not alter.

image

Figure 5. Percent change in plasma NOM, MMP-9 and serum LIGHT concentrations in morbid obese T2DM patients before and 6 months following RYGB (N = 15). Data are presented as mean ± SE * P < 0.05 by Paired t test.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Our data show clearly for the first time that the mRNA expression of IL-4, a key Th2 cytokine which modulates IgE synthesis and is involved in the pathogenesis of asthma, is significantly increased in the peripheral blood MNC of the obese and obese type 2 diabetics by ∼100% above that seen in normal subjects. Its expression is related to BMI and HOMA-IR. In addition, the expression of one other most recently described asthma related gene, LIGHT, was also increased significantly. The expression and plasma concentration of MMP-9, another important mediator of inflammation in asthma and a key factor in bronchial remodeling, were also significantly higher in the obese and the obese type 2 diabetic groups; in addition, their expression levels were related significantly to BMI and HOMA-IR. The expression of the chemokine receptor CCR2 was significantly greater in the obese and the obese diabetic and was also related to BMI and HOMA-IR. The relationship of HOMA-IR with these inflammatory mediators is of interest since our previous work has shown that insulin receptor phosphorylation in MNC is inversely related to the expression of other inflammatory mediators like NFκB binding activity, plasma CRP concentration and the expression of the suppressor of cytokine signaling-3 (SOCS-3) as well as BMI and HOMA-IR ([23]).

Because MNC fraction consists of monocytes and lymphocytes, both of which are involved in systemic inflammatory processes including those in the lung and the bronchi, it is likely that this increase of IL-4 and MMP-9 in MNC in peripheral blood cells is relevant to the pathologic processes in the bronchi and the lung. Indeed these cells are capable of infiltrating into various tissues during inflammation. It is known that broncho-alveolar macrophages participate in asthmatic inflammation and that tissue macrophages are derived from the monocytes in circulation ([24]). As IL-4, MMP-9, and LIGHT are key mediators involved in the pathogenesis of asthma, the observation of an increase in IL-4, LIGHT, and MMP-9 expression in the obese and obese type 2 diabetics is of importance and is relevant to the pathogenesis and the increased prevalence of bronchial asthma in these patients. Similarly, the increased expression of CCR-2 in these patients indicates that these cells are ready to respond through chemotaxis to key chemokines like MCP-1 and eotaxin from the sites of inflammation. MCP-1 is the major chemo-attractant for monocytes while it is also a chemo-attractant for eosinophils since they too express CCR-2. Eotaxin attracts eosinophils and also binds to CCR-2 ([25]). It is relevant that the presence of diabetes in addition to obesity did not lead to a further increase in IL-4, MMP-9, or CCR-2 expression while it did cause further increases in LIGHT expression and plasma concentrations. LIGHT plasma concentrations were shown previously to be higher only in morbidly obese and T2DM in a cross sectional study evaluating LIGHT relationship to hypertriglyceridemia ([26]).

It is equally important that following RYGB and weight loss, there was a reduction in the expression of these key genes including IL-4, MMP-9, LIGHT, LTBR, and ADAM-33. This establishes that the expression of these genes at least in part is dependent upon obesity. It is intriguing that while the expression of LTBR and ADAM-33 was not increased in the obese, RYGB led to a significant reduction in its expression. If these changes in gene expression are reflected in the clinical changes in the patient, they may provide a useful indication of the clinical activity of asthma and gastric bypass surgery may provide a potential therapeutic approach to asthma in the morbidly obese. The reduction in the expression of asthma related genes is reminiscent of the reduction in the expression of genes related to Alzheimer's disease following RYGB and weight loss that we demonstrated recently ([27]). This parallel is of interest since the risk of Alzheimer's disease is increased in the obese and since this condition is associated with chronic inflammatory changes in the brain.

It is also noteworthy that the gene most associated with asthma through genome wide scans, ADAM-33 ([17]), was not increased in the obese or the obese diabetic. There was no relationship between its expression and BMI or HOMA-IR (data not shown). It is of interest, therefore, that RYGB and weight loss resulted in a significant reduction in the expression of ADAM-33. It is thus possible that ADAM-33 expression is dependent both upon genetic factors and obesity. The expression of ADAM-33 in obese asthmatics needs to be investigated further.

The potential clinical significance of our observations is obvious. The insulin resistant proinflammatory states of obesity and T2DM are characterized by an increase in IL-4, LIGHT, MMP-9, and CCR-2 expression in MNC. There is also an increase in MMP-9 and NOM concentrations in plasma. Because these are key mediators involved in the pathogenesis of allergy and asthma, the increase in their expression and plasma concentrations may contribute to the increased vulnerability and risk of asthma in the obese. Obese T2DM patients also showed an increase in IL-4 expression when compared to normal subjects but this was no greater than that observed in the obese. It is of interest that the group with morbid obesity had the greatest increase in the expression of these genes than those with less severe obesity. There were highly significant relationships between the expression of these genes, BMI and HOMA-IR. Thus, obesity and insulin resistance may contribute significantly to the pathogenic mechanisms underlying asthma. The fact that the increase in the expression of asthma related genes was similar in the non-morbidly obese and the nonmorbidly obese type 2 diabetic patients suggests that the presence of diabetes per se does not increase the risk of asthma markedly beyond that related to obesity. It is relevant that a recent study demonstrating an increase in the prevalence of asthma in patients with T2DM showed that this increase was of the order of only 8% if the data were corrected for the concomitant presence of obesity ([2]).

There is an important limitation to this study since our investigations did not include obese patients with asthma and thus the observations cannot be applied to obese asthmatics immediately. On the other hand, this is also its strength since our data are not confounded by the presence of asthma itself. These data also point out for the first time an increase in the expression of several key genes relevant to the pathogenesis of asthma in the obese and the obese diabetic. They provide the first conceptual mechanistic link between asthma and obesity based on inflammatory mediators specifically linked to asthma.

In conclusion, patients with obesity and, in particular, those with morbid obesity and T2DM have an increase in the expression of IL-4, MMP-9, LIGHT, and CCR-2 but not ADAM-33 in MNC. The expression of these genes is related to BMI and HOMA-IR. Plasma NOM, indicative of increased iNOS activity, and MMP-9 concentrations are also elevated in the obese. Following RYGB and weight loss, there is a significant reduction in the expression of IL-4, ADAM-33, LIGHT, MMP-9, CCR-2 and plasma concentrations MMP-9 and NOM. These factors may contribute to the pathogenesis of obesity associated asthma and may be reduced after weight loss. This study should stimulate further investigations into the mechanistic aspects of obesity related asthma and into the therapeutic role of weight loss and bariatric surgery in this condition.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The authors are grateful to Dr. Stanley Schwartz (Director, Division of Allergy/Immunology/Rheumatology, State University of NY at Buffalo for critical discussions, and to Lisa Naylon, Deanne Stanton, and Jackie Diaz for clinical support, and to and to Lynne Barnas (Diabetes and Endocrinology Center of WNY, Kaleida Health) for secretarial support.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References