Hypothesis-generating analysis of the impact of non-damaging metabolic acidosis on the transcriptome of different cell types: Integrated stress response (ISR) modulation as general transcriptomic reaction to non-respiratory acidic stress?

Extracellular pH is an important parameter influencing cell function and fate. Microenvironmental acidosis accompanies different pathological situations, including inflammation, hypoxia and ischemia. Research focussed mainly on acidification of the tumour micromilieu and the possible consequences on proliferation, migration and drug resistance. Much less is known regarding the impact of microenvironmental acidosis on the transcriptome of non-tumour cells, which are exposed to local acidosis during inflammation, hypoxia, ischemia or metabolic derailment. In the present hypothesis-generating study, we investigated the transcriptional impact of extracellular acidosis on five non-tumour cell types of human and rat origin, combining RNA-Sequencing and extensive bioinformatics analyses. For this purpose, cell type-dependent acidosis resiliences and acidosis-induced transcriptional changes within these resilience ranges were determined, using 56 biological samples. The RNA-Sequencing results were used for dual differential-expression analysis (DESeq and edgeR) and, after appropriate homology mapping, Gene Ontology enrichment analysis (g:Profiler), Ingenuity Pathway Analysis (IPA®), as well as functional enrichment analysis for predicted upstream regulators, were performed. Extracellular acidosis led to substantial, yet different, quantitative transcriptional alterations in all five cell types. Our results identify the regulator of the transcriptional activity NCOA5 as the only general acidosis-responsive gene. Although we observed a species- and cell type-dominated response regarding gene expression regulation, Gene Ontology enrichment analysis and upstream regulator analysis predicted a general acidosis response pattern. Indeed, they suggested the regulation of four general acidosis-responsive cellular networks, which comprised the integrated stress response (ISR), TGF-β signalling, NFE2L2 and TP53. Future studies will have to extend the results of our bioinformatics analyses to cell biological and cell physiological validation experiments, in order to test the refined working hypothesis here.

page 2 of 4 A clean copy of the edited manuscript has been uploaded as the new manuscript file.
3. We noticed you have some minor occurrence of overlapping text with the following previous publication (s), which needs to be addressed: We compared the text of the mentioned publications (3 of the mentioned publications are from our group) with the text of our manuscript, sentence by sentence, but could not detect major overlaps.Nevertheless, we modified few sentences in the introduction that showed some similarities due to the basic and general nature of the issues described.https://journals.lww.com/jasn/pages/default.aspx  Although not specified we assume that it refers to following publication: Wesson DE, Buysse JM, Bushinsky DA (2020) Mechanisms of Metabolic Acidosis-Induced Kidney Injury in Chronic Kidney Disease.J Am Soc Nephrol 31: 469-482.The authors of this review describe, as we do in the introduction, mechanisms and cellular consequences of local acidosis.Because we were aware that similar issues are described for which the variability of wording is limited, we cited this review several times in the introduction (Ref.3).We modified few sentences in the introduction that showed some similarities due to the basic and general nature of the issues described.https://docksci.com/acidic-environment-activates-inflammatory-programs-in-fibroblasts-via-a-camp-map_5a7bbb2cd64ab22d4c439d9f.html This is a publication of our group that also deals with the impact of acidosis.Therefore, a certain overlap in wording, especially in the introduction and the method section, may occur.However, we could not detect overlaps outside the method section. This is a publication of our group that also deals with the impact of acidosis.Therefore, a certain overlap in wording, especially in the introduction especially in the introduction and the method section, may occur.However, we could not detect relevant overlaps outside the method section.Nevertheless, we modified one sentence.A. Ihling, … M. Gekle.J. Proteome Res.2015, 14, 9, 3996-4004 https://www.mdpi.com/2076-3921/12/2/412 This is a publication of our group that also deals with the impact of acidosis.Therefore, a certain overlap in wording, especially in the introduction especially in the introduction and the method section, may occur.However, we could not detect overlaps outside the method section.Ref. 13. MC Schulz, … M. Gekle.Antioxidants 2023, 12(2), 412 https://academic.oup.com/hmg/article/26/R2/R91/3978054 This is a completely unrelated publication that is unknown to us.This study deals with a neuropathological topic, amyotrophic lateral sclerosis, which is not related to our area of expertise.In their manuscript the authors discuss the general cell biological mechanism of "integrated stress response" as we do and cite a publication that we also cite (Pakos-Zebrucka K, Koryga I, Mnich K, Ljujic M, Samali A, Gorman AM (2016) The integrated stress response.EMBO rep 17: 1374-1395.).Integrated stress response is a clearly defined cellular mechanism, the characterization of which does not allow room for diverse explanations.Therefore, you may find overlapping wording whenever integrated stress response is described or discussed.However, we could not identify overlapping wording in terms of partial or even entire phrases.

We checked our revision carefully concerning the appropriate citation of all sources.
4. We note that the grant information you provided in the 'Funding Information' and 'Financial Disclosure' sections do not match.
When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the 'Funding Information' section.
We added the correct information: This project was supported funded by the Deutsche Forschungsgemeinschaft (DFG GE 905/19).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
page 3 of 4 5.We note that you have indicated that data from this study are available upon request.
This must be a misunderstanding, since we declared "Yes -all data are fully available without restriction".This is documented in the PONE-S-23-12894 file.Datasets generated and/or analyzed during the current study are available in the gene expression omnibus database.Human data: GSE220788 (token: glkxqmgclpofpiv).Rat data: GSE220789 (token: obcrcmkirbmbbgd).
6. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly.Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

Done.
7. Please review your reference list to ensure that it is complete and correct.If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references.Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript.If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:
Please respond to the comment of the reviewer to validate the finding at RT-PCR level.We performed a comprehensive bioinformatic analysis on RNA sequencing results comprising a large number of genes from five different cell types, to identify patterns and generate new hypothesis.This analysis includes bioinformatically derived upstream predictions concerning the activity of signaling modules as well as downstream predictions concerning cellular functions.These parameters cannot be validated by RT-PCR, but have to be investigated at the protein and/or functional level.These analyses are the focus of future projects and are beyond the scope of the present study.Furthermore, high quality RNA sequencing with the appropriate number of biological replicates followed by state-of-the-art differential expression analysis, including extensive quality control and normalization, leads to more reliable and valid results compared to RT-PCR with one or two housekeepers.We are convinced that the next step must be the determination of relevant alterations at the protein level (changes in expression or in modification, like phosphorylation) and/or functional cell assays.Currently we are setting up the methodology for these work packages.
The word "research" does not appear on page 9. On page 3 there is the phrase "In the past, research focused mainly on tumor microenvironment acidosis as key parameter for cell fate and disease progression."This phrase is grammatically correct.
On page 5 CO2 was changed to CO2.

Shows was changed to show.
4. I think RT-PCR experiments should be used to verify the RNS-seq data.
We performed a comprehensive bioinformatical analysis on RNA sequencing results comprising a large number of genes from five different cell types, to identify patterns and generate new hypothesis.This analysis includes bioinformatically derived upstream predictions concerning the activity of signaling modules as well as downstream predictions concerning cellular functions.These parameters cannot be validated by RT-PCR, but have to be investigated at the protein and/or functional level.These analyses are the focus of future project and are beyond the scope of the present study.Furthermore, high quality RNA sequencing with the appropriate number of biological replicates fol-lowed by state-of-the art differential expression analysis, including extensive quality control and normalization, leads to more reliable and valid results compared to RT-PCR with one or two housekeepers.We are convinced that the next step must be the determination of relevant alterations at the protein level (changes in expression or in modification, like phosphorylation) and/or functional cell assays.Currently we are setting up the methodology for these work packages.
5. The English writing should be largely improved.
Both reviewers answered question 4 above ("Is the manuscript presented in an intelligible fashion and written in standard English?") with yes.Thus, the comment raised here is contradictory.As we are experienced in the use of the English language for scientific publications (and also spend time in Australia and the US), we think we are able to evaluate a scientific text concerning appropriate English writing.Nevertheless, we went again thoroughly through the entire manuscript and checked it for grammar and spelling errors.