Autism and the Immune system – Special (Rose)

Autism Research and Treatment
Volume 2012 (2012), Article ID 986519, 10 pages
doi:10.1155/2012/986519Research Article

Intracellular and Extracellular Redox Status and Free Radical Generation in Primary Immune Cells from Children with Autism

http://www.hindawi.com/journals/aurt/2012/986519/

Shannon Rose, Stepan Melnyk, Timothy A. Trusty, Oleksandra Pavliv, Lisa Seidel, Jingyun Li, Todd Nick, and S. Jill JamesDepartment of Pediatrics, Arkansas Children’s Hospital Research Institute, University of Arkansas for Medical Sciences, Little Rock, AR 72202,  USAReceived 30 July 2011; Revised 12 August 2011; Accepted 12 September 2011Academic Editor: Antonio M. Persico Copyright © 2012 Shannon Rose et al.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The modulation of the redox microenvironment is an important regulator of immune cell activation and proliferation.

To investigate immune cell redox status in autism we quantified the intracellular glutathione redox couple (GSH/GSSG) in resting peripheral blood mononuclear cells (PBMCs), activated monocytes and CD4 T cells and the extracellular cysteine/cystine redox couple in the plasma from 43 children with autism and 41 age-matched control children. Resting PBMCs and activated monocytes from children with autism exhibited significantly higher oxidized glutathione (GSSG) and percent oxidized glutathione equivalents and decreased glutathione redox status (GSH/GSSG). In activated CD4 T cells from children with autism, the percent oxidized glutathione equivalents were similarly increased, and GSH and GSH/GSSG were decreased. In the plasma, both glutathione and cysteine redox ratios were decreased in autistic compared to control children. Consistent with decreased intracellular and extracellular redox status, generation of free radicals was significantly elevated in lymphocytes from the autistic children.

These data indicate primary immune cells from autistic children have a more oxidized intracellular and extracellular microenvironment and a deficit in glutathione-mediated redox/antioxidant capacity compared to control children.

These results suggest that the loss of glutathione redox homeostasis and chronic oxidative stress may contribute to immune dysregulation in autism.

Full study at link

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Recent studies have revealed numerous immunologic abnormalities among children with autism including alterations in immune cell proportions [37–40] and shifts in helper T-cell subpopulations after mitogenic stimulation [41, 42].

Peripheral blood mononuclear cells (PBMCs) from individuals with autism have been shown to produce higher levels of proinflammatory cytokines and abnormal levels of regulatory cytokines compared to control PBMCs at baseline and upon mitogenic stimulation [43–46].

Taken together, the immunological studies suggest a role for a dysregulated immune system in autism that potentially could be related to a deficit in glutathione-mediated antioxidant capacity and an oxidized microenvironment in immune cells.

To investigate this possibility, we examined whether primary immune cells (PBMCs) from children with autism exhibit decreased intracellular glutathione redox capacity compared to PBMCs from age-matched control children and whether a more oxidized intracellular and extracellular microenvironment is associated with increased production of oxidizing intracellular free radicals.

Because immune cells from children with autism have been shown to have abnormal responses to stimulation, we also elected to challenge the PBMCs with immune activators known to promote oxidative stress and measure the resulting intracellular glutathione redox status in activated isolated monocytes and T cells.

Discussion

Oxidative stress is generally defined as an imbalance between oxidant production and endogenous antioxidant defense mechanisms and can be clinically defined in humans by a decrease in the redox status of GSH/GSSG and cysteine/cystine thiol/disulfide redox couples [49]. The relative equilibrium between reduced and oxidized sulfhydryl groups defines the ambient redox state. Low glutathione redox status has been associated with the pathophysiology of several neurobehavioral disorders including schizophrenia [2, 50], bipolar disorder [3], alcoholism [51], HIV [52], and Alzheimer’s disease [53].

This is the first study to evaluate intracellular glutathione-mediated antioxidant/redox capacity in primary cells from children with autism as well as the extracellular plasma cysteine/cystine redox status. Because these two redox systems are compartmentalized and independently regulated, evaluation of both redox couples provides a complete picture of the primary immune cell microenvironment in children with autism. Supporting and extending our previous findings of decreased plasma and lymphoblastoid cell GSH/GSSG, we now report that both primary immune cell GSH/GSSG and plasma cysteine/cystine redox couples are similarly compromised resulting in a more oxidized immune cell microenvironment in children with autism compared to control children.

Recent evidence supports the notion that subtle fluctuations in ambient redox status may provide an important regulatory mechanism that can dynamically modulate immune cell function. Activation and proliferation of T cells require a reducing intracellular microenvironment, whereas a more oxidized environment promotes cell cycle arrest and blunted responsiveness to immune stimulation [54–57]. For example, a mechanism involving extracellular redox modulation by regulatory T cells (Tregs) was recently elucidated by Yan et al. [35]. Tregs were shown to inhibit the release of cysteine into the immune synapse between dendritic cells and naïve T cells, which effectively reduces GSH levels in T cells by eliminating the rate-limiting amino acid for GSH synthesis. A high ratio of reduced to oxidized glutathione is required for cell cycle progression from G1 to S phase and induction of the T-cell proliferative response [55]. Thus, the more oxidized GSH/GSSG redox state of the intracellular glutathione pool in PBMCs and in activated CD4 T cells observed in children with autism (Table 2) would suggest a hyporesponsive phenotype that is less conducive to T-cell activation and proliferation. Consistent with this hypothesis, several recent studies have documented abnormalities in the adaptive immune response in children with autism [44, 58].

A glutathione deficit in T cells has been shown to negatively affect the adaptive immune response and T-cell proliferation by reducing IL-2 receptor turnover and IL-2-dependent DNA synthesis [59, 60]. In monocytes, an oxidized intracellular environment has been shown to alter the cytokine profile and skew the Th1 and Th2 balance [61, 62].

Studies in mice have demonstrated that the intracellular GSH content of antigen presenting cells (APCs) reversibly alters the Th1 and Th2 cytokine response pattern [61]. Specifically, a GSH deficit reduced Th1-associated IFN-γ production and exaggerated Th2-associated IL-4 production. Restoration of GSH restored the Th1 cytokine response and normalized the Th2 response. Consistent with these observations, two independent studies have reported that helper T-cell subpopulations in PBMCs from children with autism are shifted towards T helper 2 (Th2) dominance [41, 42].

Further, a decrease in T-cell IL-2 receptor expression has been reported to be associated with decreased proliferative response after mitogen stimulation in children with autism [58].The more oxidized GSH/GSSG redox status in plasma and primary immune cells in children with autism (Figure 1) may offer a mechanistic explanation for the abnormal adaptive immune response previously reported in these children. When intracellular oxidative stress exceeds glutathione redox capacity, cells export GSSG into the plasma as a mechanism to restore internal redox homeostasis [49, 63]. The elevated GSSG concentrations in PBMCs (Table 2) suggest that the GSSG export mechanism and intracellular antioxidant capacity were not sufficient to maintain intracellular redox homeostasis and that redox imbalance was chronic in these children.

The association between a more oxidized immune cell microenvironment and an abnormal adaptive immune response warrants continued investigation especially in light of the potential reversibility of immune dysfunction with targeted treatment to restore redox homeostasis [15].The calculated values for the extracellular GSH and cysteine pools (Table 3) in our control population differ somewhat from previously published values. In adults, the plasma glutathione is more reduced at around −137 mV, and the plasma cysteine redox couple is more oxidized at −80 mV [30, 48]. These discrepancies may reflect methodological differences in sample preparation in that our electrochemical detection does not require derivatization for detection. It is also possible that children (age 3–10 years) may have less reducing capacity than previously reported in adults (age 25–35 years) [48]. Nonetheless, our calculated values are consistent with previous reports that plasma cysteine (−111 mV) is more oxidized than that of GSH (−128 mv).Mean intracellular free radical production was higher in primary lymphocytes from children with autism relative to lymphocytes from age-matched control children (Figure 2) and was driven by a subset of 5 (33.3%) children whose lymphocytes exhibited especially high levels of free radicals.

Mitochondria are the primary producers and targets of intracellular free radicals, and mitochondrial dysfunction has been postulated to be a contributing factor in the pathogenesis of autism and numerous other neurological disorders [64–67]. In a lymphoblastoid cell model, we previously demonstrated that the GSH/GSSG redox ratio in mitochondria was significantly lower in autism compared to control cells and was associated with a significantly lower mitochondrial membrane potential after nitrosative stress [16]. It is well established that mitochondria are highly concentrated in presynaptic terminals and that loss of redox control can negatively affect the efficiency of neurotransmission and synaptic plasticity [68, 69]. Similarly, mitochondrial localization and redox signaling at the immunological synapse between lymphocytes and antigen presenting cells are required for immune activation, and excessive ROS can interrupt these signaling pathways [70–72].

A recent study of mitochondrial defects in lymphocytes from children with autism found decreased complex I activity and overreplication of and deletions in mitochondrial DNA compared to control lymphocytes [73]. Based on this evidence, it is plausible to hypothesize that mitochondria may be the source of the increased levels of lymphocyte free radicals observed in the subset of autistic children presented in Figure 2. Consistent with this hypothesis, a recent meta-analysis estimated that mitochondria dysfunction may affect up to 30% of children with autism [64]. Based on this evidence, further study of mitochondrial function and redox status in lymphocytes from children with autism is warranted.Relevant to our observations, two recent papers have revealed that an oxidized extracellular cysteine/cystine redox status can initiate a redox signaling cascade that stimulates intracellular mitochondrial ROS production as a mechanism to initiate an inflammatory immune response [74, 75]. The signal transduction from the extracellular to intracellular compartments occurs through oxidative modification of redox-reactive cysteines on cell surface proteins. Exposed cysteine sulfhydryl groups on proteins can be reversibly oxidized to sulfenic acid or disulfide bonds resulting in altered protein structure and function that initiate downstream redox signaling cascades [33, 76, 77].

In an elegant series of experiments, Imhoff and Hansen demonstrated that mitochondrial ROS production was significantly increased in cells incubated under extracellular oxidized cysteine/cystine redox conditions [74]. The stimulated intracellular ROS production resulted in the expression of Nrf-2, the transcription factor responsible for initiation of the inflammatory response. Treatment to block the availability of cell surface cysteine thiol groups abrogated mitochondrial ROS production and Nrf-2 expression. Go et al. confirmed and extended these observations by demonstrating that treatment to maintain mitochondrial redox status abrogated ROS production in the presence of oxidized extracellular cysteine/cystine [75]. Although the precise mechanism for the oxidative cysteine/cystine-dependent signaling for mitochondrial ROS production is not yet clear; the authors provide evidence of a possible link to changes in the redox state of cytoskeletal proteins that could be functionally linked to the mitochondrial membrane. Other studies have demonstrated that an oxidized plasma cysteine/cystine redox potential is associated with proinflammatory conditions [78, 79] and can be modulated by diet [80, 81]. These observations support the possibility that the oxidized plasma cysteine/cystine in children with autism may be functionally related to the increase in lymphocyte free radical production observed and contribute to immune cell abnormalities in these children.

In summary, we show for the first time that both the extracellular and intracellular immune cell compartments are more oxidized in children with autism compared to age-matched unaffected control children. Randomized clinical trials will be needed to determine whether treatment to normalize plasma and intracellular redox status will improve immune cell function and possibly the health and behavior in children with autism.

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