Preprint Club
A cross-institutional Journal Club Initiative

Keywords

● Macrophages

● Inflammation

● pH sensing

Main Findings

Inflammation is a multistep process aimed a protecting the host, while eliminating invading pathogens. The acidification of the environment is one possible result of inflammation that is believed to contribute to antimicrobial activity. However, extensive acidification is suggested to negatively impact the tissue, requiring the pH to signal back to the inflammatory cells to adapt is gene expression. The authors reasoned to investigate whether alterations in environmental pH control gene expression in macrophages in response to LPS.

The authors first investigate the expression of LPS driven genes in presence of neutral or acidic pH, demonstrating that a low extracellular pH suppresses selective groups of genes while elevating others in defined kinetics. To determine whether known pH sensors expressed in macrophages modulate these kinetics the authors analysed Gpr65-/-, Gpr68-/-, Hif1a-/- and Hif2a-/- macrophages with no significant alterations in gene expression. Similarly, no pH dependent alterations in TLR signalling were observed, excluding the possibility of altered signal transduction in the regulation of gene expression. When investigating the impact of epigenetic modulation on gene expression in response to an acidic pH, the authors observed significant changes in chromatin accessibility pointing them towards proteins that may interact with histones as regulators of pH-driven gene expression.

Inspired by recent findings in budding yeast, the authors developed screening criteria for pH-dependent chromatin modulators and identified BRD4 as potential target. BRD4 formed transcriptional condensates in the nuclei of macrophages at pH 7.4. However, these condensates disappeared at pH 6.5, suggesting that BRD4’s binding to histones may be pH dependent. Using isolate nuclei from macrophages, the authors demonstrated that pH sensitive BRD4 condensates disappeared even in the absence of cell surface molecules and cytoplasm. Importantly, the formation of BRD4 condensates was dynamic and revertible once an acidic pH changed to pH 7.4. Pharmacologic inhibition of BRD4 confirmed the transcriptional condensates required BRD4 even at pH 7.4. Moreover, LPS stimulation of macrophages had the similar effect on BRD4 condensates as acidification of the pH. Importantly, these effects were also seen in human macrophages, several cell lines and primary tissue cells. Lastly, the authors demonstrated that BRD4 is actively modified by protons, which impacts the chromatin accessibility of TLR4 activated transcription factors to pH sensitive genes.

Collectively, this preprint identifies BRD4 as direct sensor of pH with significant implications in the regulation of inflammatory genes. This suggests that inflammation induced changes in the pH, which imposes secondary effects on the gene expression through the modulation of chromatin accessibility to tune inflammation by integrating the environmental pH.

Limitations

● While conducted in several cell types, the role of the described pathway in vivo in not demonstrated in report. Visualizing transcriptional condensates in vivo following LPS injection would be a phenomenal addition to this already exhaustive work.

● The authors explore the impact of LPS and TLR4 signalling in the context of distinct pH values. These findings are very important considering the relevance of this pathway but limit the breadth of its application to other inflammatory pathways. Would cytokine stimulation of fibroblasts or epithelial cells for example through IL-1R or IL-18R operate through a similar mechanism considering their convergence on NFkB? Would interferon signalling be altered in the context of distinct pHs too?

● The authors explore acidification at pH 6.5, inspired by reports centred around inflammation. However, would a higher pH value (e.g. pH 8 or 9) strengthen the transcriptional condensates?

● The authors provide evidence that most know pH sensors on macrophages are not implicated in the regulation of pH-dependent genes. However, one wonders how macrophages adapt their internal pH to a lowered externa pH. Considering that epithelial cells and fibroblasts show a response similar to macrophages the mechanism may involve a ubiquitously present pH sensor/modulator rather than a macrophage specific element.

● The authors suggest that pH-dependent genes contribute to tissue damage caused by inflammation. It would be of great advantage to demonstrate that BRD4 inhibitors improve tissue damage in vivo.

Significance/Novelty

The authors use a large array of experimental techniques and systematic approaches to provide compelling evidence for a novel mode of gene regulation that accompanies the inflammatory response. The authors findings are broadly applicable to a larger set of cells making their findings relevant to large number of researchers and areas of investigation. Their findings further provide a novel mode of gene regulation that accompanies the inflammatory response integrating the environmental acidification as feedback regulator of gene expression, independent of secondary signalling through receptor/transcription factor networks. This suggests a novel, broadly applicable, dynamic process that directs the inflammatory response.

 

Credit

Reviewed by Arthur Mortha as part of a cross-institutional journal club between the Icahn School of Medicine at Mount Sinai, the University of Oxford, the Karolinska Institute and the University of Toronto.

The author declares no conflict of interests in relation to their involvement in the review.