Poster Session - Abstract # 4

Oxidative Stress Regulator NRF2 controls Inflammatory T-helper (Th) Subset differentiation by Modulating Glycolysis

Debolina Dasgupta, Ben Choi, Kalyani Pyaram

Department of Biology, Kansas State University, Manhattan, KS, USA

Introduction

Kelch-like ECH-associated protein 1(Keap1) and nuclear factor erythroid 2–related factor 2 (Nrf2) proteins are part of the anti-oxidation pathway, which maintains redox balance in mammalian cells. Nrf2, which is usually tightly regulated by Keap1, is upregulated during oxidative stress.  It activates anti-oxidant response genes to protect cells from redox damage.  Although implicated in modulating inflammation, much remains to be understood if and how the antioxidation system affects immune responses of T-lymphocytes (T-cells).  T-cells are the specialized adaptive immune cells which orchestrate immune responses.  Upon activation, CD4 T-cells differentiate into different T-helper (Th) subsets depending upon the cytokine milieu and the threat encountered.  If the antioxidation modulates Th differentiation and/or their metabolism, is still unclear.  The goal of this study is to identify the role of Nrf2 in the differentiation of inflammatory (Th1) and regulatory (Treg) T-cell subsets and dissect the underlying metabolic mechanisms.  Future in vitro and metabolic assays will be conducted in other subsets like Th17, Th9 and Tfh to establish the role of Nrf2 in the differentiation of these subsets thereby modulating the Th cell mediated adaptive immune responses.  These studies may pave way for novel immunotherapeutic strategies.

Methods

To address this, we used mice with CD4 T-cell specific knock down of Nrf2 or Keap1.  We performed in vitro Th1 differentiation assays using Wild Type (WT), Keap1-KO (knock out) and Nrf2-KO (knock out) CD4 T-cells and assessed the expression of IFN-γ and T-bet, the signature cytokine and transcription factor for Th1, respectively.  We validated our findings by performing antigen specific activation using T- cells from OTII mice, which are specific to OVA antigen.  To dissect the metabolic mechanisms, we measured the levels of lactate, as a read out for glycolysis as Th1 cell differentiation requires glycolysis.  We also performed similar differentiation experiment in Tregs (regulatory T cells) by assessing the expression of Foxp3 which is the master transcription factor for Tregs.

Hypothesis

We hypothesize that high Nrf2 inhibits Th1 subset differentiation and promotes Treg subset differentiation by inducing a metabolic shift in CD4 T-cells.

Conclusion

We observed lower Th1 differentiation from Keap1-KO mice, indicating that high Nrf2 negatively regulates Th1 generation. Data from antigen specific activation experiments showed comparable results.  Further, lactate levels were lower in Keap1-KO CD4 T-cells than WT and Nrf2-KO CD4 T-cells suggesting that Nrf2 may inhibit glycolysis in Th1 cells.  Conversely, we observed increased Treg differentiation of Keap1-KO cells as measured by Foxp3 expression suggesting that Nrf2 promotes Treg cell differentiation.  Overall, our data indicate that Nrf2 favours anti-inflammatory status as it inhibits inflammatory subset differentiation and promotes the differentiation of the regulatory subset.