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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 1, 2004 as doi:10.1096/fj.04-2278fje. |
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* School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, UK;
Inotek Pharmaceuticals Corp., Beverly, Massachusetts, USA;
National Institutes of Health, NIAAA, Laboratory of Physiologic Studies, Rockville, Maryland, USA;
CR-UK Carcinogenesis Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK; and
|| Institute of Human Physiology and Clinical Experimental Research, Semmelweis University of Medicine, Budapest, Hungary
1Correspondence: School of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Lewes Road, Brighton BN2 4GJ, UK. E-mail: jgmabley{at}hotmail.com
SPECIFIC AIMS
8-OxoG-DNA glycosylase (OGG-1) is an enzyme involved in DNA repair. It excises 7,8-dihydro-8-oxoguanine, which is formed by oxidative damage of the DNA base guanine. The aim of this study was to investigate the role of OGG-1 in inflammation using three models of inflammation: endotoxic shock, diabetes, and contact hypersensitivity.
PRINCIPAL FINDINGS
1. OGG-1-deficient mice are resistant to systemic inflammation induced by endotoxin
Survival of male OGG-1+/+ and OGG-1–/– mice after 55 mg/kg lipopolysaccharide (LPS) i.p. was monitored over 72 h. Mortality was significantly delayed in the OGG-1–/– mice compared with OGG-1+/+ mice, with twice as many OGG-1–/– mice seen alive at 24 and 48 h as OGG-1+/+ mice (Fig. 1
A).
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Treatment of male mice with 1 mg/kg i.p. LPS for 90 min significantly increased serum levels of chemokine MIP-1
and the cytokine TNF- in OGG-1+/+ mice. LPS increased serum levels of MIP-1 and TNF- in OGG–/– mice, but this increase was markedly lower than observed in the OGG-1+/+ mice (Fig. 1B
).
Treatment of OGG-1+/+ with 80 mg/kg LPS (i.p.) for 12 h caused liver and kidney dysfunction as evidenced by increased serum levels of alanine aminotransferase, blood urea nitrogen, and creatinine (Fig. 1C
). There was an LPS-mediated increase in myeloperoxidase (MPO) activity indicative of neutrophil infiltration into the lung and heart as well as increased lipid peroxidation, indicative of oxidative stress, in the lung, heart, liver, and kidney (Fig. 1D, E
). OGG-1–/– mice exhibited a markedly reduced the degree of liver and kidney damage in response to LPS treatment vs. that observed in OGG+/+ mice. Though significantly reduced, LPS-induced neutrophil infiltration into lung and heart of OGG-1–/– mice was still observed. Oxidative stress was observed in the organs of LPS-treated OGG-1–/– mice, but again was markedly reduced in the lung and the heart compared with OGG-1+/+ mice. Surprisingly, there was no oxidative stress observed in the liver of LPS-treated OGG-1–/– mice. However, oxidative stress in the kidney of OGG-1–/– mice was similar to that observed in OGG-1+/+ mice (Fig. 1C-E
).
2. OGG-1 gene-disrupted mice are resistant to the development of diabetes
In response to multiple low-dose streptozotocin (MLDS), wild-type OGG-1 mice responded with progressive hyperglycemia and increased incidence of diabetes (Fig. 2
A, B). OGG-1–/– mice had significantly lower blood glucose levels and incidence of diabetes on day 21. The degree of insulin depletion in the pancreas of the OGG-1–/– mice was significantly attenuated compared with the response in the OGG-1+/+ mice (Fig. 2C
). The pancreatic insulin content of OGG-1+/+ mice decreased by 81% in response to MLDS treatment; in OGG-1–/– mice the decrease was only 48%, indicating that OGG-1 deficiency protects against the immune cell-mediated islet ß cell destruction.
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MLDS significantly increased pancreatic levels of the chemokine MIP-1 and Th1 cytokines TNF- and IL-12 in OGG-1+/+ mice compared with vehicle-treated mice (Fig. 2D
). MLDS treatment had no effect on pancreatic levels of the Th2 cytokines IL-4 and IL-10 in OGG-1+/+ mice. In contrast, MLDS-treated OGG-1–/– mice showed no increase in pancreatic levels of MIP-1, TNF-, or IL-12 but a dramatic increase in pancreatic levels of the Th2 cytokine IL-4 in MLDS-treated OGG-1–/– mice vs. vehicle control (Fig. 2D
). OGG-1–/– mice have higher basal levels of IL-10 in the pancreas than OGG-1+/+ mice, though there was no further increase in these levels after MLDS treatment.
3. OGG-1 gene disruption attenuates contact hypersensitivity response
Induction of contact hypersensitivity resulted in increased neutrophil accumulation in the ear tissue of OGG-1+/+ mice coupled with increased levels of the Th1 cytokines IL-1ß and TNF-, as well as chemokines MIP-1 and MIP-2 and the Th2 cytokine IL-4 compared with unsensitized controls. OGG-1–/– mice exhibited marked protection from the increases in neutrophil accumulation and exhibited an attenuation of the cytokine/chemokine responses.
CONCLUSIONS AND SIGNIFICANCE
Increased endogenous formation of reactive oxidant species is known to induce the activation of OGG-1. We have demonstrated here that disruption of the gene for the DNA repair enzyme OGG-1 protects mice and reduces the inflammatory response. In all cases the protection exerted by OGG-1 gene disruption is associated with a marked decrease in cytokine and chemokine production, as well as an improvement in organ damage. Therefore, we propose that OGG-1, in addition to being a DNA repair enzyme, functions as an inflammatory/immune system modulator.
OGG-1 is not unique in this dual function. The nuclear enzyme, poly (ADP-ribose) polymerase (PARP), overactivation in response to severe oxidant-induced DNA damage, has been shown to promote cell dysfunction, culminating in necrosis. PARP-mediated cell necrosis has been implicated as a principal form of cell and organ damage in various forms of reperfusion injury. PARP is linked to promotion of cell recruitment, inducing organ injury in inflammation. This effect on immune cell recruitment has been linked to a costimulatory effect of PARP on production of chemokines MIP-1 and MIP-2, and its modulatory effect on a variety of transcription factors including NF-
B, AP-1, and STAT-1. It is conceivable that OGG-1 plays a role similar to PARP in regulating proinflammatory gene transcription. It remains to be determined whether it is the basal activity of OGG-1 modulates the inflammatory response or, alternatively, it is the catalytic activation of OGG-1 in response to DNA damage in response to endogenous oxidant generation during inflammation.
Could OGG-1’s protective effect be mediated through an inhibition of PARP activation? OGG-1 functions as a DNA repair enzyme via removal of 8-oxoG. It then cleaves DNA, leaving a single-strand break, which is subsequently repaired by other follow-up repair enzymes. As single-stranded DNA breaks result in activation of PARP, it is conceivable that normal function of OGG-1 could be associated with activation of PARP in situations associated with increased cellular oxidative stress. LPS activates the immune system by interacting with the TLR-4 receptor, leading to subsequent transcription factor activation, cytokine and chemokine production, resulting in oxidative stress-mediated organ dysfunction and subsequent death. The OGG-1–/– mouse is extremely resistant to most of these LPS-induced effects. A massive degree of oxidative stress and cellular damage is observed after LPS administration that induces a variety of DNA modifications, including DNA single-strand breakage and PARP activation. Given the presence of multiple triggers of PARP activation in endotoxic shock, removal of OGG-1-mediated activation of PARP would be expected to reduce cellular PARP activation only to a small degree and is unlikely to be the principal mechanism of protection.
A possible mechanism of immunosuppression in OGG–/– mice may be to modulate cytokine production. Data from all three inflammation models demonstrate that in OGG–/– mice, levels of chemokines and destructive Th1 cytokines are reduced.
In conclusion, the present results demonstrate that OGG-1 is a novel regulator of the immune system (Fig. 3
) and appears to be involved in a variety of pathophysiological conditions, including autoimmunity and allergy. OGG-1 may emerge as a target for pharmaceutical intervention in experimental therapy of allergy and inflammation.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2278fje;
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