Compartmentalized redox regulation is a key component of redox signaling and regulation of cell functions. Studies by Curran and coworkers showed that early stress response by AP-1 involved redox regulation in cell nuclei 
. DNA binding was inhibited by oxidation of Fos and Jun, and Trx1 reversed this inhibition in the presence of NADPH, Trx1 reductase, and a nuclear redox factor (Ref-1). Ref-1 was found to be an activity of a DNA repair enzyme apurinic/apyrimidinic endonuclease 
. Subsequent studies showed that similar nuclear redox control mechanisms function for NF-κB, Nrf-2, GR, estrogen receptor, p53 and HIF-1α 
. Importantly, Hirota et al. found that cytoplasmic Trx1 suppressed NF-κB activation by UV irradiation, whereas targeted expression of Trx1 in nuclei enhanced NF-κB activity by increasing DNA binding 
. These results clearly discriminated nuclear and cytoplasmic redox events in transcriptional regulation. Site-directed mutagenesis suggested that lysine residues near the C-terminus of Trx1 provide a nuclear targeting sequence 
. Together, the studies show that in response to oxidative stress, nuclear Trx1 functions to maintain redox-sensitive transcription factors in reduced, DNA-binding forms.
Nuclear Trx1 is relatively reduced under non-stressed conditions 
and highly resistant to oxidation compared to cytoplasmic Trx1, mitochondrial Trx2 and cellular GSH 
. Thus, there would not appear to be a need for nuclear translocation in the absence of oxidative challenge. None-the-less, Trx1 is found in nuclei of cells at growth boundaries 
. This indicates that nuclear translocation of Trx1 occurs in response to signals other than overt oxidant stress. Nuclear targeting of Prx1, a Trx1-dependent peroxidase, enhances NF-κB reporter activity while cytoplasmic targeting of Prx1 blocks this activity 
, suggesting the existence of a constitutive H2
-dependent mechanism to inhibit transcriptional activity. H2
production within nuclei occurs due to oxidative demethylation of histones 
, and stimulation of oxidant production appears to be a common event associated with DNA damage and repair 
. Thus, the pattern of nuclear Trx1 in growth boundaries and the increase in response to stress may reflect temporal sequences in which transcriptional activation is followed by a nuclear oxidation that coordinately decreases activity of redox-sensitive transcription factors. Together, these observations suggest that nuclear Trx1 has dual functions in supporting activation of redox-sensitive transcription factors and also in inhibiting inactivation mechanisms that depend upon endogenous oxidants.
A model incorporating these concepts to account for excessive immune response due to increased nuclear Trx1 is summarized in . The response to infection/inflammation includes: 1) decreased GSH levels by oxidative stress; 2) NF-κB activation and nuclear translocation; 3) elevation of NF-κB activity in nuclei due to increased abundance of Trx1 (NLS-hTrx1) and reduction via Ref-1; 4) increased cytokine gene expression by NF-κB and enhanced immune response; 5) decreased oxidative inactivation of NF-κB due to enhanced elimination of nuclear H2
by Trx1-dependent Prx-1 and Prx-2; 6) excessive stimulation of NF-κB activity; and 7) increased morbidity and mortality from excessive immune response. NF-κB activation is induced by viruses/viral products as well as other stimuli associated with oxidative stress (free radicals, UV light, gamma-irradiation) and could be generally relevant to conditions with increased signaling by inflammatory cytokines IL-6 
and TNFα 
. Responses to infection-induced inflammatory stimuli include feedback stimulation, e.g. NF-κB activation stimulates inflammatory cytokine up regulations. The response is dependent upon GSH so the redox regulation may be more complex than implied by this model.
A scheme for stimulation of inflammatory response in NLS-hTrx1 Tg by infection.
In the absence of viral challenge, the NLS-hTrx1 mice did not show gross phenotypic differences, e.g., weight, growth, fecundity, appearance, activity, from WT, and no major effects were observed in other thiol antioxidant proteins. This suggests that the present model can be useful to study oxidative processes that occur in cell nuclei. In this regard, the model adds to the growing number of mouse models useful to translate compartment-specific mechanistic information to in vivo
. This nuclear redox model may also be useful in combination with other compartmental redox models (e.g., Trx2 Tg mouse, 
to provide tools to help discriminate between toxicological stimuli targeting nuclear redox functions from those targeting cytoplasmic and mitochondrial functions. The HeLa cells studies (), showed that increased NF-κB luciferase activity due to expression of NLS-hTrx1 (same vector as used for Tg mouse) was blocked by the dominant negative NLS-C35S-hTrx1. Consequently, development of mouse models with dominant negative Trx1 or an NF-κB reporter may enhance further understanding of redox control system in cell nuclei in vivo
The exacerbation of injury with increased nuclear Trx1 is distinct from previous Trx1- and Trx2-transgenic mouse studies that showed protection against injury 
. This distinct characteristic is reflected in the GSH data for lung and plasma (), which showed that the increased nuclear Trx1 did not affect cellular GSH or redox potential changes in response to infection but caused a significant downstream decrease in plasma GSH and oxidation of plasma Eh
GSSG. This is consistent with evidence that reductive stress, as well as oxidative stress, can contribute to disease mechanisms due to disruption of redox signaling and control. For instance, recent studies show involvement of reductive stress in cardiomyopathy 
. High levels of Hsp27 in transgenic mice resulted in cardiac hypertrophy in association with decreased ROS level, and significant increase in GSH content and the ratio of GSH/GSSG 
. A consequence of reductive stress resulting from Hsp27 over-expression was a significantly reduced survival rate 
. Rajasekaran et al. also demonstrated that cardiomyopathy induced by mutation in human αB-crystallin was under reductive stress due to elevated levels of glutathione peroxidase, increased GSH content and the ratio of GSH/GSSG and γ-glutamylcysteine synthetase 
Contribution of nuclear Trx1 to hyperactivity of the immune system could provide the basis for therapeutic development to inhibit nuclear Trx1 translocation as a means to prevent excessive activation following influenza infection. With some capacity to maintain reduction of transcription factors already present in nuclei, controlling further increase might limit intensity of activation without blocking necessary activity. Therapeutics targeting Trx1 translocation may also be useful for adult respiratory distress and multi-systems organ failure where post-infection immune responses contribute to tissue injury and death 
. This approach could be useful to indirectly control stress-induced glucocorticoid responses, or in cancer therapeutics, provide a novel mechanism to control HIF-1α activity 
In summary, the present studies with H1N1 influenza viral infection in NLS-hTrx1 Tg mice show that increased nuclear Trx1 enhances activity of redox-sensitive transcription by NF-κB, causes exaggerated immune response and contributes to disease severity. Consistent with this response, redox-sensitive glucocorticoid-induced cell death was also exacerbated in thymocytes from Tg compared to WT littermates. Together, the results show that increased nuclear Trx1 has a critical role in stimulating intensity of immune responses. This suggests that nuclear translocation of Trx1 may be a useful therapeutic target to prevent severity of disease caused by excessive or prolonged activation of redox-sensitive transcription factors.