Activation of the transcription factor NRF2 mediates the anti-inflammatory properties of a subset of over-the-counter and prescription NSAIDs

Abstract

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase (COX) enzymes and are ubiquitously used for their anti-inflammatory properties. However, COX inhibition alone fails to explain numerous clinical outcomes of NSAID usage. Screening commonly used NSAIDs in primary human and murine myeloid cells demonstrated that NSAIDs could be differentiated by their ability to induce growth/differentiation factor 15 (GDF15), independent of COX specificity. Using genetic and pharmacologic approaches, NSAID-mediated GDF15 induction was dependent on the activation of nuclear factor erythroid 2-related factor 2 (NRF2) in myeloid cells. Sensing by Cysteine 151 of the NRF2 chaperone, Kelch-like ECH-associated protein 1 (KEAP1) was required for NSAID activation of NRF2 and subsequent anti-inflammatory effects both in vitro and in vivo. Myeloid-specific deletion of NRF2 abolished NSAID-mediated tissue protection in murine models of gout and endotoxemia. This highlights a noncanonical NRF2-dependent mechanism of action for the anti-inflammatory activity of a subset of commonly used NSAIDs.

Keywords: growth/differentiation factor 15; nonsteroidal anti-inflammatory drugs; nuclear factor erythroid 2-related factor 2.

Figure 1.. Indomethacin induces GDF15 independent of cyclooxygenases.

(A) Gdf15 mRNA expression in BMDMs following stimulation with 0.5 mM indomethacin (n=3 per group) and (B) at 6 hours following stimulation with increasing doses of aspirin (0.25mM, 0.5 mM, 1 mM), indomethacin (0.125mM, 0.25mM, 0.5mM), ibuprofen (0.125mM, 0.25mM, 0.5mM), ketoprofen (0.25 mM, 0.5mM, 1mM), naproxen (0.25 mM, 0.5mM, 1mM), etodolac (0.25 mM, 0.5mM, 1mM) and rofecoxib (0.25 mM, 0.5mM, 1mM), in order of increasing specificity of COX2 as compared to COX1 (n=4 per group). (C) Supernatant GDF15 protein levels following 24 hours stimulation with vehicle, 0.5 mM indomethacin and 0.5 mM ketoprofen (n=5–8 per group). (D) GDF15 mRNA expression in human MDM following stimulation by 0.5 mM indomethacin and 0.5 mM ketoprofen (n=3 per group). € Gdf15 mRNA expression of BMDMs stimulated for 6 hours with vehicle or 0.5 mM indomethacin (n=4 per group). (F) Supernatant GDF15 protein levels from BMDMs stimulated for 24 hours with vehicle or 0.5 mM indomethacin (n=4 per group). All experiments are representative and repeated at least once. Statistics by ANOVA. Data are represented as mean ± standard deviation. *P<.05, ***P<.001, ****P<.0001, ns P>.05

Figure 2.. A subset of NSAIDs activate NRF2 and downstream GDF15.

(A) Gdf15 mRNA expression from BMDMs derived from B6, Myd88^−/−, Chop^−/−, Nrf2^−/−and Eif2a^−/− mice treated for 6h with vehicle or 0.5 mM indomethacin (n=4 per group). (B) Supernatant GDF15 protein levels from BMDMs treated for 24h with vehicle, 0.5 mM indomethacin or 0.5 mM ketoprofen (n=4 per group). (C) Gdf15 mRNA expression from BMDMs treated for 6h with vehicle, sulforaphane or dimethylfumarate (DMF) (n=5–6 per group). (D) Supernatant GDF15 protein levels from BMDMs treated for 24h with vehicle or DMF (n=4 per group). Messenger RNA expression of Cat, Gar, Gclm, Hmox1, Nqo1, Taldo1 from BMDMs derived from (E) B6 and Nrf2^−/− mice (n=4 per group) or (F) Ptgs1/2 f/f mice following stimulation with vehicle or 0.5 mM indomethacin for 6h (n=4 per group). (G) Immunofluorescence staining of BMDMs for NRF2 (green) and DAPI (blue) following treatment with vehicle or indomethacin. (H) Chromatin immunoprecipitation of Gdf15 and Nqo1. ChIP was performed on BMDM stimulated for four hours with vehicle or indomethacin using anti-NRF2 (top, red), anti-H3K27Ac (bottom, black) or control (IgG) antibody and qPCR was performed with primers spanning 1 kb upstream and downstream of the identified ARE consensus sequence (Fig. S3) in the regulatory elements of Gdf15 (left) and Nqo1 (right). Representative of 2 independent experiments. All experiments are representative and repeated at least once. Statistics by ANOVA. Data are represented as mean ± standard deviation. *P<.05, **P<0.01, ***P<.001, ****P<.0001, ns P>.05

Figure 3.. Indomethacin activates NRF2 target genes, including GDF15, in NRF2-depdendent, and COX1/2-independent manner, in Lyz2-expressing cells in vivo.

(A) Baseline GDF15 serum levels (n=3–10 per group). (B) Serum GDF15 protein levels in B6 mice following intraperitoneal injection with vehicle, 15 mg/kg indomethacin or 15 mg/kg ketoprofen (n=3–5 per group). Serum GDF15 protein levels in (C) Nrf2^−/− (D) Nrf2 f/f and (E) Ptgs1/2 f/f mice following intraperitoneal injection of 15 mg/kg indomethacin (n=3–6 per group). (F) Serum GDF15 protein levels following injection of 25 mg/kg sulforaphane (n=3–7 per group). (G) Hmox1 RNA expression of bulk liver in animal treated with vehicle or indomethacin at the indicated time points (n=3–4 per group). Kidney Gdf15, Hmox1, and Nqo1 mRNA expression 6h following intraperitoneal injection of 15 mg/kg indomethacin or 50 mg/kg sulforaphane in (H) Nrf f/f mice (n=3–6 per group) or (I) Ptgs1/2 f/f mice (n=5–6 per group). All experiments are representative and repeated at least once. Statistics by ANOVA. Data are represented as mean ± standard deviation. *P<.05, **P<0.01, ***P<.001, ****P<.0001, ns P>.05

Figure 4.. Cysteine 151 of KEAP1 is required for NRF2 activation by indomethacin.

(A) Gdf15 mRNA levels in BMDM generated from C151S KEAP1 transgenic animals and littermate controls 6 hours after stimulation with indomethacin or ketoprofen (n=4 per group). (B) NRF2 target gene expression in BMDM generated from C151S KEAP1 transgenic animals and littermate controls 6 hours after stimulation with indomethacin (n=4 per group). (C) Baseline GDF15 serum levels in C151S KEAP1 mice and controls (n=5–7 per group). (D) Plasma GDF15 levels after an intraperitoneal injection of 15 mg/kg indomethacin at the indicated time points in C151S and wildtype animals (n=5–7 per group). (E) mRNA expression of Gdf15, Hmox1, and Nqo1 at 6 hours from bulk liver of C151S and wildtype animals after treatment with 15 mg/kg indomethacin (n=4 per group). All experiments are representative and repeated at least once. Statistics by ANOVA. Data are represented as mean ± standard deviation. **P<0.01, ***P<.001, ****P<.0001

Figure 5.. Indomethacin reduces inflammation in endotoxemia and gout via an NRF2-dependent, GDF15-independent and COX1/2-independent mechanism.

(A) Temperature curve for B6 mice following intraperitoneal injection with LPS and vehicle, indomethacin or ketoprofen (AUC p <0.0001, (n=4 per group). Temperature curve for (B) Nrf2 f/f mice (AUC p<0.0085, n=5 per group), (C) C151S mice (AUC<0.001, n=5 per group), and (D) in B6 or Nrf2^−/− mice treated with vehicle or indomethacin with or without recombinant GDF15 or protein control (BSA) (AUC p<0.01, n=4–5 per group) (E) Serum IL-1ß levels in B6 and Nrf2^−/− mice 24 hours following intraperitoneal injection of LPS and vehicle or indomethacin (n=4–5 per group). (F) Log2 fold change in mRNA expression of inflammatory markers in the liver of B6 and Nrf2^−/− mice injected intraperitoneally with LPS and vehicle or indomethacin (n=3–5 per group). (G) Serum alanine transferase (ALT) activity 20 hours following intraperitoneal injection with LPS and vehicle or indomethacin (n=4–5 per group). (H) Liver Nqo1 and Cat mRNA expression following intraperitoneal injection of vehicle, indomethacin or ketoprofen (n=3–5 per group). Footpads of (I) Ptgs1/2 f/f mice (n=3–4 per group) and (J) Nrf2 f/f mice (n=4–8 per group) injected intradermally with PBS or MSU and then treated with intraperitoneal injections of vehicle or indomethacin. Foot diameter was normalized to baseline foot diameter. All experiments are representative and repeated at least once. Statistics by ANOVA. Data are represented as mean ± standard deviation. *P<.05, **P<0.01, ***P<.001, ****P<.0001, ns P>.05