The understanding of how macrophages regulate their response to toxic particles via two independent pathways could uncover therapeutic opportunities to quell inflammation.Human exposure to toxic particles drives various diseases. Examples include gout, an acute arthritis driven by monosodium urate crystals, or MSUc; CPPD disease, another inflammatory joint disease driven by calcium pyrophosphate dihydrate crystals, or CPPDc; and the lung disease silicosis, driven by inhaled silica-derived nanoparticles.
Macrophages are the specialized phagocytic cells that respond to toxic particle depositions to exert inflammatory and clearance responses. These long-lived immune cells engulf foreign substances like toxic particles, as well as pathogens like cancer cells and microbes, and cell debris to defend against infection and injury. Digestion takes place in intracellular macrophage organelles called lysosomes.
A hallmark of phagocyte activation in gout inflammation is ingestion of MSUc followed by rupture of the macrophage’s phagolysosome.
In research published in the journal Immunity, an international team of 30 scientists show that particle uptake by macrophages elicits two separate transcriptional pathways to differentially activate a unique inflammatory and lysosomal gene expression program. Knowledge of how the macrophage controls these separate gene expression programs during particle-induced inflammation could uncover therapeutic opportunities to modify the destructive inflammation from encounters with toxic particles, says Isidoro Cobo, Ph.D., of the University of Alabama at Birmingham, who co-led this research with Christopher Glass, M.D., Ph.D., University of California, San Diego.
“While particle recognition and phagocytosis are well understood, the epigenetic and transcriptional mechanisms regulating these signaling pathways remain incompletely defined,” Cobo said. “Identifying transcription factor networks in macrophages, their upstream pathways and modulators of downstream effectors could uncover therapeutic opportunities to modify inflammation triggered by particle exposure and deposition.”
Researchers performed a systematic analysis of the initial transcriptomic responses of macrophages to four different particles. They exposed mouse macrophages to MSUc, CPPD, crystalline silica particles and aluminum salts for five hours, and then performed RNA sequencing. Between 841 and 2,275 genes were upregulated by exposure to one of the types of particles, and between 781 and 1,835 genes were downregulated.
Of the upregulated genes, 313 were upregulated by all the four particles, and those common genes were enriched for inflammatory signaling and hydrolase activity, especially the acidification of the lysosome that enables its function. The four particles also caused lysosomal damage. There were also 131 in-common downregulated genes. Thus, there were both particle-specific effects on gene expression and a shared program of up- and downregulated genes for all the particles.
Isidoro Cobo, Ph.D.In experiments with both mouse and human macrophages, the researchers showed that common lysosomal acidification gene response was regulated by a novel AMPK-TFEB/TFE3-DNMT3A/DOT1L axis. In contrast to lysosomal acidification genes, the common inflammatory gene response of cytokine and chemokine expression was regulated independently by a previously demonstrated JNK kinase-AP-1 transcription factor pathway. This reveals a bifurcation between inflammatory and lysosomal responses to particles.
For the lysosomal acidification gene response, AMPK is a kinase that activates other proteins through phosphorylation. TFEB and TFE3 are transcription factors that regulate gene expression by binding to specific DNA sequences to turn genes on or off. The researchers showed regulation by TFEB and TFE3 by analyzing genome-wide binding of the two transcription factors in macrophages stimulated with MSUc. They found that the genes exclusively bound by these transcription factors were involved in lysosomal function, including acidification. This role of TFEB and TFE3 was further confirmed by gain- and loss-of-function experiments.
DNMT3A methylates DNA at specific targets to control gene expression. DOT1L methylates the lysine 79 residue of histone H3, one of the histone proteins that help package and organize DNA within the nucleus. Changes in histone methylation also can alter the accessibility of genes to be expressed.
Researchers found that increased DOTL1 activity was required for the activation of the lysosome acidification program by particles. They also found that DNMT3A transcriptional activation by TFEB and DOT1L was required for lysosomal gene expression and acidification of macrophages stimulated with particles. Of note, these results are independent of DNMT3A’s catalytic function in DNA methylation, indicating an alternative role of DNTMTA in regulating gene expression. Proximity ligation-assisted chromatin immunoprecipitation, a method to identify long-range chromatin interactions, was used to help understand this mechanism of DNMT3A gene activation.
Finally, investigation of which signaling pathway acts upstream of TFEB to regulate lysosomal acidification gene expression in macrophages during response to particles showed that AMPK signaling was required for the activation of the lysosomal program of macrophages during stimulation by MSUc.
“Our model therefore proposes that DOT1L and DNMT3A act as epigenetic regulators that direct TFEB and TFE3 toward lysosomal acidification programs while AP-1 regulates inflammatory genes, establishing distinct transcriptional networks in response to particle-induced macrophage activation,” said Cobo, an assistant professor in the UAB Department of Medicine Division of Clinical Immunology and Rheumatology. “This dual regulatory mechanism, defined by JNK and AMPK signaling and reinforced by the selective recruitment of DNMT3A and DOT1L, offers insights into how macrophages regulate lysosomal integrity and inflammatory responses independently.”
The Immunity study led by Cobo and Glass, “Particle uptake by macrophages triggers bifurcated transcriptional pathways that differentially regulate inflammation and lysosomal gene expression,” has 28 co-authors from the UAB Department of Medicine Division of Clinical Immunology and Rheumatology; the University of California, San Diego School of Medicine; the Veterans Administration San Diego Healthcare System, San Diego, California; Washington University School of Medicine, Saint Louis, Missouri; the University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; the Medical University of Vienna, Vienna, Austria; the Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain; the Dana-Farber Cancer Institute, Boston, Massachusetts; and the Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
At UAB, Medicine is a department in the Marnix E. Heersink School of Medicine, and Cobo is a scientist in the Comprehensive Arthritis, Musculoskeletal, Bone and Autoimmunity Center.