Particle uptake by macrophages triggers bifurcated transcriptional pathways that differentially regulate inflammation and lysosomal gene expression.

Exposure to physical particles is a driver of several inflammatory diseases. Here, we systematically investigated responses of macrophages to pathogenic forms of monosodium urate crystals, calcium pyrophosphate crystals, aluminium salts, and silica nanoparticles. While each particle induced a distinct pattern of gene expression, we also identified a common signature that was preferentially linked to inflammation and acute activation of genes required for lysosomal acidification. Using monosodium urate crystals as a model system, we obtained evidence that the program of lysosomal gene expression was regulated by a network of transcription factors that included TFEB and TFE3 and the epigenetic regulators DNMT3A and DOT1L. This lysosomal acidification network operated in parallel with, but largely independently of, a JNK and AP-1 dependent transcription factor network that drove crystal induced chemokine and cytokine gene expression. Mechanistically, the uptake of particles led to early activation of AMPK signalling that was required for activation of TFEB and TFE3 in a process independent of reduced mTOR signalling. These results thereby revealed a mechanism by which the functions of TFEB and TFE3 can be preferentially targeted to specific aspects of lysosomal gene expression, which may provide insights into therapeutic approaches to treat individuals with particle-associated diseases. Overall design: We performed histone acetylation analysis by ChIP-Seq of the enhancer landscape of macrophages exposed to particles. We used RNA-Seq to undestand the underlying transcriptional response. We used ChIP-Seq for certain transcription factors to understand the underlying molecular mechanisms. Flushed bone marrow from both femurs and tibias of C57BL/6, Tfeb-Tg, Bhlhe40-/- and Bhlhe41-/- mice were cultured in DMEM medium supplemented with 10% de-complemented FBS, Penicillin-Streptomycin 100 U/mL and 16 ng/mL of recombinant murine M-CSF (Peprotech, #315-02). Fresh M-CSF was added 72h after seeding. Ampka1-/- BMDM were generated as explained elsewhere (Wang et al. 2016). Briefly, bone marrows from 7-week-old to 8-week-old Ampka1-/- and congenic WT mice were cultured in complete Roswell Park Memorial Institute (RPMI) media containing 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 µg/mL) in the presence of 5 ng/mL of MCSF (R&D Systems). On day six after seeding, BMDM were scrapped out and plated one day prior treatment with Metformin (MP Biomedicals, #151691), 125 mM A769662 (Sigma, # SML2578), or since day 0 with 1 µM SGC0946 (Selleckchem, #S7079). BMDM were then treated with control PBS or 250 µg/mL of MSUc, calcium pyrophosphate dihydrate crystals, silica nanoparticles (Invivogen, #tlrl-sio-2), or aluminum hydroxide salt (Invivogen, #tlrl-aloh) at various timepoints as explained in the text and Figure legends.