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[email protected] Recent Patents on Anti-Infective Drug Discovery, 2016, 11, 000-000
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RESEARCH ARTICLE
Complement 3 Receptor Expression in Individuals with Type 2 Diabetes Judy Ly1, Devin Morris1,2, Minette Lagman1,2, Christopher Ng1, Shelby Anderson2,#, John Daliva2,#, Naji Muwanas2,#, Igal Tarash2,#, Cesar Ochoa3, Airani Sathananthan2,3 and Vishwanath Venketaraman1,2* 1
Graduate College of Biomedical Sciences, Western University of Health Sciences, Pomona, CA 91766; 2College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, Pomona, CA 91766; 3Western Diabetes Institute, Patient Care Center, Western University of Health Sciences, Pomona, CA 91766
ARTICLEHISTORY Received: February 27, 2016 Revised: May 23, 2016 Accepted: June 2, 2016 DOI: 10.2174/1574891X11666160608121 617
Abstract: Background: According to the World Health Organization, as of 2014 9% of the world’s adult population is affected by diabetes. Uncontrolled diabetes is a pro-inflammatory process that increases generation of reactive oxygen species (ROS).
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Methods: The production of ROS leads to a chronic increase in oxidative size should be 4" x 4" inches stress which results in an increased susceptibility to infections. Individuals with type 2 diabetes mellitus (T2DM) are highly susceptible to Mycobacterium tuberculosis (M. tb) infection. Previous research has demonstrated that Vishwanath Venketaraman glutathione (GSH) plays an important role in the control of M. tb infection. Recent studies have demonstrated that phagocytosis of M.tb is diminished in patients with T2DM. Phagocytosis in macrophages is thought to be mediated in part by complement protein 3b (C3b)-complement protein receptor 3b (C3R) interactions. Since C3b production is not diminished in patients with T2DM we propose that C3R production is reduced and is the cause for impaired macrophage phagocytosis as well as IL-12 and IFN- signaling. Conclusion: This study utilizes a quantitative PCR (qPCR), demonstrating decreased transcription of C3R mRNA in patients with T2DM as compared to non-diabetics.
Keywords: Diabetes, tuberculosis, glutathione, complement receptors, immune responses. INTRODUCTION Currently the World Health Organization reports that an estimated 347 million people worldwide have diabetes. It is expected that by 2030 the number of individuals with diabetes will increase by 50% each year. T2DM accounts for 90-95% of all diabetes [1]. Unfortunately, diabetic patients not only suffer from diabetes-related complications but are also at a higher risk for being infected with M. tb, the causative agent of tuberculosis *Address correspondence to this author at the Microbiology/Immunology, Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, 309 E Second Street, Pomona, CA 91766-1854; Tel: 909-706-3736; E-mail:
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(TB). Diabetic patients have a two to threefold higher risk of contracting TB compared to healthy individuals [2, 3]. Additionally, diabetic patients suffering from active TB have a higher risk of death during TB treatment [4, 5]. Previous research has demonstrated that glutathione (GSH) plays an important role in the control of M. tb infection by its direct antimycobacterial effects and by enhancing the functions of immune cells that contribute to innate and adaptive immune responses against M. tb infection [6-14]. GSH is present in two forms; the active form, reduced glutathione (rGSH) and the inactive oxidized glutathione (GSSG). The primary role of rGSH is to neutralize ROS; rGSH is consumed in this process and converted to GSSG. Uncontrolled © 2016 Bentham Science Publishers
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diabetes is a pro-inflammatory process that increases generation of ROS, leading to a chronic increase in oxidative stress and depletion of intracellular rGSH which results in an increased susceptibility to infections [6-13]. In addition, our previous findings suggest that GSH levels in T2DM patients are compromised not only due to oxidative stress but due to diminished levels of enzymes that are responsible for the synthesis of GSH [6-13]. Phagocytosis in macrophages is thought to be mediated in part by complement protein 3b (C3b)complement protein receptor 3b (C3R) interactions. C3R is an opsonin dependent receptor. C3R binds to particles that are marked with iC3b, a portion of C3R. Those particles that express iC3b will be marked for phagocytosis by C3R. Additionally, further phagocytosis will not occur until an additional stimulus is received [15, 16]. This stimulus will enhance C3R’s response to the particle that is now bound resulting in phagocytosis. Cytokines are one of the stimuli that triggers this final response for phagocytosis [15, 16]. In addition to controlling phagocytosis, C3R signaling has been shown to control production of Interleukin-12 (IL12) and Interferon- (IFN-), two cytokines that are key in controlling intracellular infections such as M. tb [13, 17]. While phagocytosis is impaired in diabetics, C3b levels in the plasma are higher than those in non-diabetics [18]. It has also been demonstrated that complement proteins (C3, C3a, C3b & Ficolin-3) are overexpressed in patients with diabetes compared to healthy controls [19]. Therefore, impaired diabetic phagocytic activity cannot be ascribed to an absence of C3b protein. However, C3R levels have not thus far been quantified in diabetics and may be the cause for the diminished phagocytic activity seen in individuals with T2DM. Furthermore, signaling through C3R regulates the production of IL-12, which stimulates the production of IFN- through Th1 response. IFN- can enhance the antimicrobial effector mechanisms inside the macrophages to leading to control of intracellular M. tb infection. Previous research shows that both IL-12 and IFN- levels are diminished in serum of diabetic patients compared to healthy individuals [3, 13, 18]. Additionally, there is compromised production of IL-12 and IFN- by PBMCs (peripheral blood mononuclear cells) from diabetic individuals [19, 20].
Ly et al.
It is our prediction that C3R expression is reduced in individuals with T2DM leading to impaired macrophage phagocytosis as well as decreased production of IL-12 and IFN- (Fig. 1 and 8). We tested our prediction by performing in vitro studies using isolated monocytes and plasma derived from healthy subjects and individuals with T2DM. Our study findings indicate that there is decreased transcription of C3R mRNA in patients with T2DM as compared to nondiabetics. MATERIALS AND METHODS Study Participants This study was approved by the Western University of Health Sciences Institutional Review Board. A total of 10 volunteers (5 T2DM patients, and 5 non-diabetic controls) were recruited for the study. The participants ranged from age 25 to 65 years old. Non-diabetic control criteria included healthy liver function and HbA1c level 5.1 - 5.9. T2DM criteria included participants with HbA1c > 9 and were not taking either Biguanide or Thiazolidinedione therapy. From our previous studies, we know that participants with HbA1c > 9 consistently demonstrated compromised levels of GSH. We therefore recruited subjects with HbA1c > 9 for this study so that we can establish a correlation between decreased GSH and downregulation in the expression of C3R in T2DM. Separation of Blood Components Approximately 30 mL of blood was drawn from all participants. Density gradient centrifugation with Ficoll-Paque was used to separate plasma, red blood cells, and peripheral blood mononuclear cells (PBMCs) from the whole blood. Isolation of Monocytes Monocytes were isolated from the PBMCs of healthy and T2DM patients as follows: PBMCs from the study subjects were washed three times with PBS, resuspended in RPMI with L – glutamine, HEPES (Corning Cellgro, 1-041-CM) and 5% human AB serum, plated on a 96 well tissue culture plates pre-coated with 0.005% poly-Llysine, and incubated overnight at 37 °C to allow monocyte adherence. After overnight incubation, nonadherent cells were removed. Fresh RPMI was added to adherent monocytes.
Complement 3 Receptor Expression in Individuals with Type 2 Diabetes
Healthy
Recent Patents on Anti-Infective Drug Discovery, 2016, Vol. 11, No. 2
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GSH Measurements GSH measurement was done using the GSH detection kit from Arbor Assays. Both total GSH and oxidized GSH were measured in monocyte lysates of both categories according to our previously published protocol [22, 23]. Malondialdehyde (MDA) Measurements
T2DM
MDA, a byproduct of lipid peroxidation, is used as a measurement of oxidative stress. MDA levels were measured in monocyte lysates of both healthy and T2DM samples. MDA was measured using a TBARS Assay Kit. The manufacturer’s protocol was followed to obtain results. Concentrations measured were corrected for protein levels. Gene Expression Measurement (RT-PCR and qPCR)
Fig. (1). Model describing compromised expression of C3R in individuals with T2DM.
Using the RNA collected from the samples, reverse transcriptase PCR (RT-PCR) was used to reverse transcribe to complementary DNA (cDNA). Quantitative PCR (qPCR) was performed on the cDNA samples to determine expression of complement receptor 3 (C3Rb) relative to an internal control gene GAPDH.
Monocyte Infection Studies
Statistical Analysis
All infection studies were performed using the virulent laboratory strain of M. tb, H37Rv inside the biosafety level 3 (BSL-3) facility. Monocytes were infected with processed H37Rv at a multiplicity of infection of 10:1. Infected cultures were treated with either 5mM N-acetyl cysteine (NAC), 10mM NAC, 5μM liposomal glutathione (LGSH), 10μM LGSH, or 10 mM Malic Acid (MA, GSH chelator). RNA was collected from infected macrophages terminated at one hour and five-days post-infection using Trizol Reagent (Life Technologies).
Data was analyzed using Graph Pad Prism Software. The levels of cytokines were compared between healthy individuals and those with T2DM, using an unpaired T- test with Welch’s correction. Reported values are means +/- standard error at a p
