Antioxidants in excess can increase inflammation and blunt benefits of exercise
Antioxidants, even glutathione, taken in excess can increase rather than ameliorate harmful inflammation as attested by two revealing papers recently published in PLOS (Public Library of Science). In a fascinating study on experimental colitis, the authors demonstrate that certain levels of ROS (reactive oxygen species, well known to be damaging at higher levels) are critically necessary to regulate the Treg (regulatory T cell) function that controls autoimmune inflammation. Moreover, of premiere clinical importance is their observation that higher levels of glutathione that excessively dampened ROS blocked Treg function and thus worsened the inflammation of inflammatory bowel disease (IBD). Antioxidants, of course, are employed to keep the lid on ROS. And with good reason:
"Reactive oxygen species (ROS) are highly reactive and interact with many bio-molecules. At high concentrations, they are likely to destroy biological structures, promoting cellular damage and tissue destruction. Traditionally, ROS have been implicated in ageing and the progression of inflammatory and autoimmune diseases, including inflammatory bowel diseases (IBD).
However...
Meanwhile, many recent observations are opposing the traditional concept on ROS, suggesting the protective role of ROS in immune-mediated inflammatory diseases."
The authors relate the fascinating background to this study on ROS, antioxidants and autoimmune inflammation:
"Mice with lower level of ROS than WT mice due to defects in ROS-producing enzyme system, such as Ncf1−/− or Nox2−/−, are more susceptible to autoimmune diseases, such as arthritis and encephalomyelitis. Humans with lower levels ROS than normal persons, such as chronic granulomatous disease (CGD) patients and carriers, are also more susceptible to autoimmune diseases. By contrast, mice with higher level ROS than WT mice due to the defect in a ROS metabolizing enzyme, glutathione peroxidase-1 (GPx-1), are resistant to immune-mediated inflammatory diseases, such as allergen-induced airway inflammation and high fat diet-induced atherosclerosis. In particular, mice with higher level of ROS due to defect of a non-enzymatic cellular anti-oxidant, peroxiredoxin (Prx) II, are resistant to dextran sodium sulfate (DSS)-induced colitis."
In other words, more enzyme activity producing antioxidants such as glutathione and peroxiredoxin increased susceptibility to autoimmune inflammatory disorders. Moreover...
"These clinical or experimental observations implicated the immunoregulatory role of ROS, and adoptive-transfer of CD4+ cells from rats with lower ROS level induced arthritis in rats with normal ROS level, demonstrating the key role of CD4+ cells in the hyperinflammatory response in lowered levels of ROS. On the other hand, oxidative stress induces T cell hyporesponsiveness in several human pathologies (e.g. cancer, rheumatoid arthritis, AIDS and leprosy). Accordingly, ROS level is supposed to be closely associated with T cell responsiveness. In particular, regulatory T cell (Treg) function seems to be closely linked to ROS level. Tregs isolated from mice with lower level of ROS, such as Ncf1−/− mice, were hypofunctional than WT Tregs. Tregs were also hypofunctional in vitro at lowered levels of ROS by adding antioxidants or NADPH oxidase inhibitors. Differentiation of inducible Treg (iTreg) seems also linked to the level of ROS. Induction of FoxP3+ iTreg was attenuated, whereas that of Th17 cells was enhanced in lowered levels of ROS due to Nox2 deficiency or addition of apocynin. By contrast, induction of FoxP3+ Treg was enhanced in elevated levels of ROS due to PrxII deficiency."
Thus, whereas excessive oxidative stress (ROS) with insufficient antioxidants can contribute to T cell hypoactivity in some pathologies including cancer and AIDS, levels of regulatory T cell (FoxP3+ iTreg) that rein in autoimmune Th17 inflammation are suppressed in the presence to ROS levels that are too low.
So the authors set out to investigate the suppressive function of Tregs isolated from mice with elevated levels of ROS due to defects in the enzymes that make the antioxidatns glutathione (GPx1) and catalase. Their results illuminated the aspects of ROS and antioxidants that are critical to case management of inflammatory disorders:
"In the present study, we demonstrated for the first time that Tregs were hyperfunctional in elevated level of ROS by using GPx1−/− × Cat−/− Tregs. As it has been already reported that Tregs were hypofunctional in lowered levels of ROS, it could be argued that Treg function is closely linked to ROS level. Actually in the present study, IP injection of NAC [N-acetylcysteine] into GPx1−/− × Cat−/− mice reduced the suppressive function of Tregs to the level comparable to WT Tregs. Administration of NAC also has made GPx1−/− × Cat−/− mice, which are naturally resistant, susceptible to DSS-induced colitis, suggesting the critical role of ROS in the prevention of DSS-induced colitis. The importance of Tregs in the maintenance of intestinal immune balance has been already shown in many other studies. Consequently, ROS level might be critical in the maintenance of intestinal immune homeostasis, providing an insight for the immunomodulation by ROS."
In other words, elevated ROS increase the antiinflammatory activity of Treg cells, while administration of NAC (which increases glutathione production) suppressed Tregs by reducing ROS, resulting in increased intestinal inflammation.Clinical note: Excessive suppression of ROS by overenthusiastic application of antioxidants can disturb immune homeostasis resulting in increased autoimmune inflammation.The authors also shed light on the association of ROS and inflammation with tryptophan metabolism:
At molecular level, the expression of an immunoregulatory enzyme, IDO, is also associated with ROS level. IDO catabolizes the essential amino acid tryptophan into the stable metabolite, kynurenine. Consequently, IDO depletes tryptophan from the environment, thus starving effector cells. It was also found that tryptophan depletion resulted in inhibition of Th17 cell differentiation and expansion of Foxp3+ Tregs....Thus, IDO expression might be induced as a consequence of the inflammatory reaction, contributing to the feedback regulation...Elevated levels of ROS not only contribute to the induction but also enhance the enzyme activity of IDO, as superoxide radical acts as a cofactor of IDO. Therefore, high expression and strong activity of IDO from the beginning in GPx1−/− × Cat−/− mice might contribute to the preparation of immunosuppressive environment preventing inflammatory tissue damage during treatment with DSS."
Clinical note: antioxidant and ROS status can be investigated with a urinary organic acid analysis that includes p-Hydroxyphenyllactate, 8-Hydroxy-2'-deoxyguanosine, kynurenate and quinolinate; and a blood assay for oxidative stress that includes cysteine, cystine, glutathione, glutathione peroxidase, lipid peroxides, sulfate, superoxide dismutase (SOD), and total antioxidant capacity (TAC).The authors summarize implications for modulation of ROS by antioxidants:
"Actually in the present study, the frequency of FoxP3+ cells was significantly increased in parallel with significantly attenuated inflammatory reaction in the lesions of DSS-induced colitis in mice with elevated level of ROS due to defects in GPx1 and Cat. By contrast, IP injection of NAC significantly reduced the frequency of FoxP3+ cells and aggravated inflammatory reaction in the lesions of DSS-induced colitis... we demonstrated an experimental colitis was attenuated in elevated level of ROS. Enhancement of Treg function and IDO expression, investigated in the present study, might be involved in the underlying mechanism...Taken together, the results of the present study suggest the potential therapeutic strategy for IBD through immunomodulation by ROS."
ROS and Psoriasis
The same team of scientists a study also in PLOS One similarly investigating the role of ROS and by implication the use of antioxidants in regard to psoriatic dermatitis:
"Psoriasis is a chronic inflammatory skin disease resulting from immune dysregulation. Regulatory T cells (Tregs) are important in the prevention of psoriasis. Traditionally, reactive oxygen species (ROS) are known to be implicated in the progression of inflammatory diseases, including psoriasis, but many recent studies suggested the protective role of ROS in immune-mediated diseases. In particular, severe cases of psoriasis vulgaris have been reported to be successfully treated by hyperbaric oxygen therapy (HBOT), which raises tissue level of ROS. Also it was reported that Treg function was closely associated with ROS level. However, it has been only investigated in lowered levels of ROS so far."
The authors state:
"Psoriasis is known to develop as a result of immune dysregulation, in particular hyperfunction of T helper 17 (Th17) cells. In steady state, immune homeostasis is maintained by regulatory T cells (Tregs) that suppress immune effectors including Th17 cells. It was also reported that psoriasis is associated with impaired suppressive function of Tregs. Therefore, in order to restore the dysregulated immune status in psoriasis, it is necessary to suppress immune effectors including Th17 cells and/or to enhance Tregs."
Consonant with their study on colitis:
"Traditionally, reactive oxygen species (ROS) are known to be implicated in the progression of many inflammatory diseases. As ROS are highly reactive and interact with many bio-molecules, they are likely to destroy biological structures, promoting cellular damage and tissue destruction. In contrast, many recent evidences are accumulating on the protective role of ROS in immune-mediated diseases. Autoimmune arthritis was aggravated in rodents with lower levels of ROS than wildtype (WT) mice due to defects in ROS-producing enzyme system, such as mutation in the neutrophil cytosolic factor (Ncf)-1 or NADPH oxidase (NOX)2. In human, too, many autoimmune diseases develop more frequently in chronic granulomatous disease (CGD) patients with lower level of ROS than normal persons due to defect in ROS-producing NOX. To the contrary, experimentally induced asthmatic inflammation was attenuated in mice with higher level of ROS than WT mice due to the defect of a ROS metabolizing enzyme, glutathione peroxidase-1 (GPx-1). Atherosclerotic lesions induced by high-fat diet were also decreased in GPx-1−/− mice. In addition, experimental colitis was attenuated in mice with a higher level of ROS due to defect in a non-enzymatic anti-oxidant, peroxiredoxin II."
So they set out to...
"...clarify the relationship between ROS level and Treg function, as well as their role in the pathogenesis of psoriasis, we investigated imiquimod-induced psoriatic dermatitis (PD) in association with Treg function both in elevated and lowered levels of ROS by using knockout mice, such as glutathione peroxidase-1−/− and neutrophil cytosolic factor-1−/− mice, as well as by using HBOT or chemicals, such as 2,3-dimethoxy-1,4-naphthoquinone and N-acetylcysteine.
Here too they found that moderately high levels of ROS were protective against autoimmune inflammation PD (psoriatic dermatitis):
"The results consistently showed Tregs were hyperfunctional in elevated levels of ROS, whereas hypofunctional in lowered levels of ROS. In addition, imiquimod-induced PD was attenuated in elevated levels of ROS, whereas aggravated in lowered levels of ROS. For the molecular mechanism that may link ROS level and Treg function, we investigated the expression of an immunoregulatory enzyme, indoleamine 2,3-dioxygenase (IDO) which is induced by ROS, in PD lesions. Taken together, it was implied that appropriately elevated levels of ROS might prevent psoriasis through enhancing IDO expression and Treg function."
Reflecting on the clinical significance of these results and the crucial difference between moderately high levels of ROS which are protective versus higher levels that are damaging:
"In the present study, we demonstrated that imiquimod-induced PD was attenuated in elevated levels of ROS, whereas aggravated in lowered levels of ROS. This observation provides experimental evidence supporting the immunoregulatory role of ROS, that is contradictory to the traditional concept. Traditionally, ROS is implicated in the progression of inflammatory diseases by promoting cellular damage and tissue destruction as well as ageing. At the moment, it is necessary to establish a new conceptual framework where the recent observations and the traditional concept can be compromised...Taken together, we can imagine a threshold level of ROS that divides the moderately high tolerable range and the intolerably higher levels. In the higher levels such as GPx-1−/−×GPx-2−/− mice, inflammatory reactions are augmented due to the direct tissue damage by ROS. Many evident previous observations that contribute to establish the traditional concept of ROS, such as vascular reperfusion injury and other in vitro observations, might fall in this range of intolerably higher levels of ROS. In contrast, as demonstrated previously by others and by us in the present study, inflammatory diseases are attenuated in the moderately high tolerably ranges such as in GPx-1−/− or PrxII−/− mice. Thus, we suppose some kinds of anti-inflammatory mechanisms are operating in the moderately high tolerable range of ROS. As ROS can induce direct tissue damages at high levels, it would be natural to develop defensive or compensatory mechanisms counteracting the destructive effects of ROS in the body."
Clinical note: Here is where the rubber meets the road in clinical case management of autoimmune diseases in respect to ROS versus excessive use of antioxidants:
"In this aspect, enhancement of Treg function depending on ROS level is quite pertinent to counteract the destructive damages induced by ROS, as Tregs suppress every arm of immune response, including Th1, Th2, Th17, B, NK cells and DCs...Tregs play a critical role in the prevention of autoimmune diseases, and functional impairment of Tregs is important in the pathogenesis of psoriasis. Therefore, restoration or strengthening of impaired Treg function would be a desirable therapeutic strategy for psoriasis. The results of the present study suggested appropriately elevated levels of ROS could enhance Treg function, and thus might attenuate psoriasis."
In particular for clinicians involved in case management of autoimmune disorders, from a personal communication with one of the lead authors Ju-young Seoh, MD, Ph,D.:
"Your understanding, “too much as well as too little antioxidant activity can be harmful, and too aggressive use of antioxidant agent, such as vitamin C, could impair Treg activity and accelerate autoimmune inflammatory activity.” is exact our message."
The authors of a paper just published in Clinica Chimica Acta also note the distinction between low and high levels of ROS and the role of antioxidants:
"Oxidative stress plays a pivotal role in the development of human diseases. Reactive oxygen species (ROS) that includes hydrogen peroxide, hyphochlorus acid, superoxide anion, singlet oxygen, lipid peroxides, hypochlorite and hydroxyl radical are involved in growth, differentiation, progression and death of the cell. They can react with membrane lipids, nucleic acids, proteins, enzymes and other small molecules. Low concentrations of ROS has an indispensable role in intracellular signalling and defence against pathogens, while, higher amounts of ROS play a role in number of human diseases, including arthritis, cancer, diabetes, atherosclerosis, ischemia, failures in immunity and endocrine functions. Antioxidants presumably act as safeguard against the accumulation of ROS and their elimination from the system."
Antioxidants and Exercise
It's edifying in this context to consider the effects of antioxidants on desirable physiological adaptations to the stress of exercise. In a study recently published in the Journal of the International Society of Sports Nutrition the investigators documented that while antioxidants increased the strength of muscle contraction acutely, there was also a suppression of the desirable growth hormone (GH) response to exercise (which increases muscle mass).
"Antioxidant supplementation is known to increase human endogenous antioxidant (AOX) capacity providing a means of blunting exercise induced reactive oxygen species (ROS). The purpose of this study was to compare the effects of a single acute dose of an AOX (vs blinded placebo) on muscle contractile performance and hormonal responses to a single bout of lower limb ‘hypertrophic’ resistance training (RT)."
Their data should be considered in the context of antioxidant supplementation and exercise:
"It was found that in comparison to a placebo mixture, subjects were able to perform 3.75% more work (W), and generate greater mean concentric power and velocity throughout the HTS after consuming the AOX mixture...however circulating GH levels was significantly reduced in the AOX trial compared to the placebo trial...This would suggest that the GH results from this study indicate they may be undesirable in regards to promoting muscular hypertrophy. It is therefore of interest for future studies to examine whether this decreased circulating GH would affect muscular hypertrophy after a prolonged period of use or whether it acutely affects IGF-1 levels. Moreover, recent research suggests excessive AOX supplementation may hinder important physiological training adaptations. This has prompted the suggestion that optimal oxidant content for maximal force production exists within the muscle...GH secretion is involved in MH and strength development and its attenuation may negatively impact training adaptations...These recent findings and the GH results in this study, highlight the need to further our understanding of the effect of AOX supplementation on training adaptations."
These results further validate the findings documented in paper published a couple of years earlier in the journal Sports Medicine:
"High levels of reactive oxygen species (ROS) produced in skeletal muscle during exercise have been associated with muscle damage and impaired muscle function. Supporting endogenous defence systems with additional oral doses of antioxidants has received much attention as a noninvasive strategy to prevent or reduce oxidative stress, decrease muscle damage and improve exercise performance...The consistent finding is that antioxidant supplementation attenuates exercise-induced oxidative stress. However, any physiological implications of this have yet to be consistently demonstrated, with most studies reporting no effects on exercise- induced muscle damage and performance. Moreover, a growing body of evidence indicates detrimental effects of antioxidant supplementation on the health and performance benefits of exercise training. Indeed, although ROS are associated with harmful biological events, they are also essential to the development and optimal function of every cell. The aim of this review is to present and discuss 23 studies that have shown that antioxidant supplementation interferes with exercise training-induced adaptations."
Here again we see that antioxidants have both positive and negative effects:
"The main findings of these studies are that, in certain situations, loading the cell with high doses of antioxidants leads to a blunting of the positive effects of exercise training and interferes with important ROS-mediated physiological processes, such as vasodilation and insulin signalling. More research is needed to produce evidence-based guidelines regarding the use of antioxidant supplementation during exercise training."
Bottom line: The use of antioxidants must be calibrated with careful consideration of the balance between protective and suppressive effects according to the needs of the individual patient by observing appropriate lab values for ROS and oxidative damage, outcomes for regulation of inflammation, and the patient's subjective impression of energy versus fatigue.