Cytokine Levels in Plasma Samples of Individuals with HIV Infection

Research Article

Austin J Clin Immunol. 2014;1(1): 1003.

Cytokine Levels in Plasma Samples of Individuals with HIV Infection

Enrique Vera Tudela1, Manpreet Kaur Singh1, Minette Lagman1, Judy Ly2, Nishita Patel3, Cesar Ochoa3 and Vishwanath Venketaraman1, 2,*

1Graduate College of Biomedical Sciences, Western University of Health Sciences, Pomona, CA 91766, USA

2Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, Pomona, CA 91766-1854, USA

3Western Diabetes Institute, Pomona, CA 91766, USA

*Corresponding author: Vishwanath Venketaraman, Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, 309 East Second Street, Pomona, CA 91766-1854, USA

Received: January 04, 2014; Accepted: January 30 , 2014; Published: February 06, 2014


CD4+ T helper cells play a vital role in the immune system by secreting cytokines, which regulate the immune response. Cytokines are secreted by T cells when an intracellular infection is detected as in case of HIV infection. HIV is known to infect T cells that have CD4+ receptors present on their surface. These cells, among others in the immune system, secrete cytokines. The levels of cytokines present in the plasma become an indicator of the nature of the immune response. In this study we are measuring levels of plasma cytokines in HIV infected individuals and healthy control subjects using ELISA. Our results indicate that cytokine plasma levels between the two groups are substantially different. In general, we observed that there was an increase in anti-inflammatory cytokines and a decrease in pro-inflammatory cytokines in individuals that were positive for HIV infection, likely due to the role of HIV in suppressing the appropriate immune response in HIV positive individuals.


Having claimed more than 36 million lives so far and with approximately 35.3 million people infected with HIV as of October 2013, HIV is a major global health issue [1]. The Human Immunodeficiency Virus (HIV) targets the immune system and weakens the surveillance and defense system of the body againstinfections, resulting in HIV infected individuals becoming more susceptible to a wide range of infections normally cleared by the immune system of a healthy individual [1].

Cytokine levels are indicative of the nature of the immune response in a particular individual. When immune cells detect intracellular pathogens, the immune response that results is specifically tailored to optimize the clearance of the pathogen [2]. T helper cells, also named CD4+ T helper cells (Th), are immune cells with an important role in directing the response necessary to accomplish this task. When the response calls for the clearance of an intracellular pathogen, a Th1 response is induced [2]. On the contrary, when removal of an extracellular pathogen is necessary, the resultant Th2 response is induced [2]. In contrast to the Th1 response, the Th2 response mediates antibody production [3]. Cytokines generally can be divided into 2 major categories, the pro-inflammatory cytokines, which stimulates the immune system, or the anti-inflammatory cytokines which suppresses the immune system [2]. Not only are CD4+ T helper (Th) lymphocytes crucial regulators of the immune system, but they also play a major role in inflammatory disease progression. One of the most important factors in directing CD4+ T helper cells towards the appropriate response are cytokines produced by cells of the innate immune system [4]. Antigen presenting cells (APC) are innate cells that activate T helper cells, causing them to differentiate into effector cells specialized in cytokine secretion and function. Th1 activation also requires interleukin 12 (IL-12) [5]. IL-12 is secreted by a class of APC called dendritic cells (DC). IL-12 induces native T-cells into the Th1 (CD4+) subset. Th1 cells then go onto produce cytokines IL-2 and interferon gamma (IFN-γ). IFN-γ acts by activating macrophages and is essential for eliminating intracellular pathogens [6]. Interleukin 2 (IL-2) is crucial for growth, proliferation and differentiation of T-cells, acting as a growth factor for T-cells [3].

Studies conducted earlier have revealed that HIV-infected individuals have a weaker immune system; this is due to the inability of CD4+ T cells to proliferate, due to the decrease in the levels of IL-12 [7]. A consequence of this decrease in IL-12 results in a decrease in IL-2 and IFN-γ, leading to immunosuppressive effects and allowing room for opportunistic infections, which is a trademark of HIV progression. This shows how CD4+ T cells play a central role in disease progression [8]. CD4 T cell counts are important biomarkers for AIDS progression in HIV patients [9]. A range of 500-1000 cells/ mm3 is considered a normal range for healthy individuals, while a CD4 count of 200 cells/mm3 is diagnostic for AIDS [10].

Given HIV’s role in dampening the immune response towards intracellular pathogens, pro-inflammatory cytokines should be decreased in HIV infected individuals, while the opposite effect would be true of anti-inflammatory cytokines. Levels of anti-inflammatory cytokines interleukin 10 (IL-10), for example, should be relatively decreased in healthy individuals with intracellular infections, allowing the infection to be better cleared by the appropriate Th1 response. Likewise, Transforming Growth Factor Beta (TGF-ß), a cytokine with an important role in augmenting a regulatory T cell response (T-regs) [11] that inhibits the immune system, should be decreased in healthy individuals with intracellular infection. Another cytokine, interleukin 6 (IL-6), has been found to serve as a growth factor for the HIV [12]. Because HIV infection weakens the cell mediated immune response and directs it to allow for its own survival, we would expect levels of IL-10 and TGF-β to be elevated in HIV-infected subjects. IL-6, serving its role as a growth factor for HIV, should also be elevated in HIV infection, as compared to healthy individuals.

In our controlled study, we aim to shed light on various components of the innate and adaptive immune system, and subsets within adaptive immunity such as the Th1, Th2, and regulatory signaling in the context of HIV infection. In light of previous findings, we hypothesize that IFN-γ, IL-12, and IL-2 will be decreased in the plasma of HIV infected subjects, while IL-6, IL-10, and TGF-β will be elevated in such subjects.

Methods and Materials

Study Participants

A total of 17 individuals were recruited to participate in this study. The age of study subjects ranged between 18-65. Of the 17 individuals, ten individuals were healthy and seven were positive for HIV infection. All of the study participants were volunteers that were local to Pomona or the nearby areas and were recruited using email, flyers at the Patient Care Center at Western University of Health Sciences in Pomona, California. The purpose of the study and what was required from each participant was explained to each individual before obtaining their consent to be subjects in our study. Background information for the HIV infected individual, such as time since seroconversion and CD4 T helper cell counts was obtained.

Collection of blood and separation of plasma sample

On day one, all of the study participants had about 40CCs of blood collected using Vanutainer Safety-Lok blood collection set. Plasma was separated from the whole blood by density gradient centrifugation using ficoll hypaque, followed by allocation into tubes and storage at -80 degrees. This plasma was then tested for cytokine levels using Enzyme-linked Immunosorbent Assay (ELISA).

Plasma cytokine measurements

Cytokine levels were measured using a Ready-Set-Go! ELISA kit from ebioscience. A total of six cytokines were measured in the plasma of all subjects. The ebioscience protocol outlined in each kit was followed. All the standard safety precautions were taken at all times.

Statistical Analysis

Graph Pad Prism 6 was used to perform statistical analysis on our data. To test for a statistically significant result, we analyzed the cytokine plasma concentrations using the Mann-Whitney test of unpaired t test data.


HIV positive participants in this study had CD4 T cell counts below the normal range of 500-1000 cells/mm3. The HIV positive participants consisted of two females and five males between the ages of between 28 and 53 (Table I). Overall, we did not observe a significant correlation among age and sex and CD4 counts. Likewise, duration of HIV treatment did not appear to affect the results of this study. There was a statistically significant difference in the plasma cytokine concentration between HIV positive and healthy individuals, as assayed with ELISA. Furthermore, those cytokines that strengthen the immune response to HIV infection such as IL-12 (Figures 2a and 2b), IL-2 (Figures 3a and 3b), and IFN-γ (Figures 1a and 1b), were significantly lower in HIV positive participants. On the other hand, cytokines that encourage HIV replication and proliferation, IL-6 (Figures 4a and 4b), IL-10 (Figures 5a and 5b), and TGF-β (Figures 6a and 6b), were significantly increased in participants positive for HIV infection. Overall, homeostasis between pro-inflammatory and anti-inflammatory cytokines was not observed. Additionally, there was a wide variation between the participants in plasma CD4 count, possibly in result to previous treatment or lifestyle habits of the participants.