Nuclear factor of activated T cells (NFAT) is a regulator of macrophage nitric oxide production in response to bacterial and viral motifs

Introductions: Immune cells, including macrophages, are activated by pathogen associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs), including Toll like receptors (TLRs) to express inducible NO synthase (iNOS), as part of the innate immune response. Activation of TLRs results in engagement of downstream transcription factors including NF-κB. A distant relative of NF-κB is the calcium/calcineurin-dependent transcription factor nuclear factor of activated T-cells (NFAT). NFAT is a key transcription factor in adaptive immunity and crucial for initiating immune responses in T cells. However, whilst the role of NF-κB is well studied, the role of NFAT in innate immune responses and inflammation is relatively unexplored (1). Recent studies have implicated NFAT in the production of pro-inflammatory cytokines downstream of TLR signalling in macrophages (2) although effects on iNOS responses were not addressed. Here we have investigated the role of NFAT, versus NF-κB, in iNOS responses to a range of TLR agonists.

Methods: J774.1 mouse macrophages, cultured in standard DMEM supplemented with 2% FCS, were pre-treated (30 minutes) with the NF-κB inhibitor BMS-345541 (BMS), cyclosporineA (CsA), which inhibits NFAT and other pathways, or VIVIT, a specific NFAT blocker, before being stimulated with bacterial (TLR4, LPS:TLR1/2, Pam3CSK4: TLR2/6, FSL-1) or viral(TLR3, Poly(I:C)) PAMPs for 24 hours. iNOS activity was measured by NO release using the Griess assay.

Results: All TLR agonists induced iNOS activity, which was inhibited by CsA, VIVIT or BMS (Fig 1). In the presence of the highest concentration of BMS used, CsA acted in an additive manner to inhibit iNOS activity (Fig 2).

Conclusion: Our data demonstrate that NFAT regulates iNOS activity induced by both bacterial and viral PAMPs where it acts in parallel to NF-κB signalling. This work suggests that NFAT is not only a critical pathway in adaptive immunity but also in innate immune responses to both bacteria and viruses. As previously proposed for NF-κB, NFAT may therefore also represent a therapeutic target for the treatment of inflammatory diseases where TLRs and iNOS have been implicated. Future experiments, using techniques that can detect nitric oxide in a more sensitive manner, will establish if NFAT regulates iNOS activity also in human macrophages.

Figure 1. Effect of NFAT or NF-?B inhibition on iNOS activity in mouse macrophages stimulated with bacterial and viral PAMPs. J774.1 cells were pre-treated with CsA (3 µM), VIVIT (10 µM) or BMS-345541 (3 µM) and stimulated LPS, Pam3 and FSL-1 (all 0.1 µg/ml) or Poly(I:C) (10 µg/ml) PAMPs for 24 hours. Data are mean ± SEM; *p<0.05 vs vehicle by Student’s t-test.

Figure 2. Effect of BMS and CsA alone and in combination on iNOS activity. J774.1 cellsweretreatedwithindicated concentration BMS and CsA and stimulated with LPS as in Fig. 1. Data are mean ± SEM.

(1) Fric J et al. (2012) Blood 120: 1380-9

(2) Minematsu H et al. (2011) Cell Signal 11: 1785-93