HISTONE DEACETYLASE 7 IN MURINE GRAM-NEGATIVE ACUTE LUNG INJURY

Reply to the Letter to the Editor: We appreciate the insightful comments on the above letter to the Editor, that discusses how our findings (similar to our previously published work on the topic)^1,2 may contradict those of prior studies on Trichostatin A (TSA) inhibition of the Histone Deacetylase (HDAC) complex.³ We also want to thank the Editor for giving us the opportunity to respond to the Letter.

Citing a research letter by Finnin et al,⁴ one of our manuscript readers posits that TSA exerts its HDAC inhibitory effect by directly binding to the HDAC catalytic region, and hence should not have any effect on HDAC7 expression. However, the cited manuscript merely assessed the TSA ability to bind to a bacterial HDAC homologue (a protein that shares only 35.2% of the amino acid sequence of human and murine HDAC1, as clearly pointed out in the referenced manuscript) in vitro, not its effect on HDAC expression. Therefore, an argument that TSA does not alter HDAC7 expression cannot be made from the referenced article, as this topic is not even explored in the cited manuscript.

The reader also states that there are no papers to his knowledge describing the effect of TSA on HDAC expression. Not only are there too numerous to cite in a Scientific Letter manuscripts that have studied HDAC expression after TSA administration, but one of the more prominent ones is cited by the reader himself: In that article, Yagi et al demonstrate that HDAC9 and HDAC11 expression decrease after TSA administration.⁵ That HDAC11 is downregulated with TSA in the Yagi manuscript, is consistent with our findings, despite the different models of Acute Lung Injury used between Yagi’s manuscript (two-hit viral and bacterial model) and ours (gram negative bacterial pneumonia), and the different timepoints (36 hours in the Yagi manuscript vs. 6 hours in ours) selected for assessment.

The reader states that we claim that the ‘E. Coli effects is due, at least in part, to the changes in HDAC7 expression’, while a closer examination of our manuscript reveals no such claim. It is also stated that the effects of HDAC7 siRNA and TSA cannot be found on HDAC7 expression, while those are clearly demonstrated, not only for HDAC7, but for all 1–11 HDACS and Sirt1 in Figure 4. We want to thank the author for pointing out a typographical error that we inadvertently made, however. The statement “This transcription is inhibited further with both TSA and highly selective HDAC7-siRNA inhibition” should read “This transcription is inhibited further with TSA administration”.

The reader also ‘assumes that the authors used an excess of siRNA to efficiently inhibit HDAC7 production’ to refute our hypothesis of HDAC7 mRNA restoration due to rapid turnover, while we clearly state that ‘we demonstrated >40% inhibition of quantifiable HDAC7 protein with HDAC7-siRNA’ (Figure 5). We also state in the Discussion that we were satisfied with this low-level HDAC7 inhibition in that this effect may be more clinically relevant and simulating real-life pharmacologic inhibition, as opposed to near zero protein levels. A statement that ‘any siRNA designed to decrease expression of specific mRNA, but not protein, therefore, it is unclear, how the protein level of HDAC7 decreased without decreasing HDAC7 mRNA level’ is irrelevant to our project, as we specifically aimed to limit HDAC7 protein translation, as this is the enzymatically active form of HDAC7 (mRNA is an enzymatically inactive nucleotide, functioning merely as a guide for the HDAC7 protein synthesis). We also propose a hypothesis for this finding, which we are currently actively testing. We agree that the finding of HDAC7 mRNA and protein levels moving in opposite directions in the face of infection is highly interesting, just like our reader, and propose potential mechanisms for this, and aim to study this phenomenon further in the near future.

Lastly, the reader observes that with TSA and HDAC7 siRNA not having the same effects on both mRNA AND protein synthesis, the conclusion that TSA acts mainly through HDAC7 inhibition cannot be made. We also want to clarify that nowhere in the manuscript is such a claim made, rather HDAC7 inhibition is key to in the regulation of the early inflammatory response that leads to Acute Lung Injury after gram-negative pneumonia. It is also important to reiterate that only the protein is believed to be enzymatically active in our clinical relevant scenario, with the mRNA merely encoding for it, and its direction of regulation is likely irrelevant, as long as the protein levels expressed remain the same.