Silencing of long isoforms of nuclear factor erythroid 2 like 1 primes macrophages towards M1 polarization.

Macrophages are a major component of the immune system and play an important role in regulating the magnitude, duration, and quality of the inflammatory response. Dissecting the functions of transcription factors regulating macrophage activation is important for understanding the inflammatory responses. Nuclear factor erythroid 2 like 1 (NFE2L1, also known as Nrf1) is a CNC-bZIP protein, which has multiple isoforms. While the exact physiological functions of various isoforms of NFE2L1 are still under investigation, accumulating evidence indicate that long isoforms of NFE2L1 (NFE2L1(L)) are important regulators in the antioxidant response, proteasome homeostasis and inflammation. In this study, we found that NFE2L1(L) was upregulated in response to LPS stimulation in RAW264.7 macrophages. Stable knockdown of Nfe2l1(L) (Nfe2l1(L)-KD) in RAW264.7 cells resulted in increased expression of multiple genes indicative of M1 polarization, including Il6, Il1β, Cox2, and Ccl2, under both resting and LPS-challenged conditions. In addition, lentiviral shRNA-mediated silencing of NFE2L1(L) in human monocytic SC and THP1 cells also significantly increased mRNA expression of IL6, IL1β, and TNFα. Furthermore, transient silence of NFE2L1(L) in primary human monocytes isolated from peripheral blood by nucleofection with small interfering RNA resulted in increased expression of IL6 and TNFα. Analysis of the key transcription factors involved in M1 polarization revealed that Nfe2l1(L)-KD RAW264.7 cells have increased mRNA and protein expression and phosphorylation of STAT1 and STAT3 under both resting and M1 polarized conditions. Activation of the NFκB, ERK1/2 and p38 pathways in response to LPS was not affected by the reduction of NFE2L1(L). Moreover, Nfe2l1(L)-KD cells were found to have elevated levels of intracellular ROS, but macrophage M1 polarization induced by Nfe2l1(L) silence was independent of ROS accumulation. Collectively, our results show that knockdown of Nfe2l1(L) leads macrophages to M1 polarization by disinhibition of STAT1/3, and not through the NFκB, ERK1/2 and/or p38 signaling pathways. These findings indicate that NFE2L1(L) functions as a negative regulator of M1 polarization and pro-inflammatory response in macrophages.