Re-programming of GM-CSF-dependent alveolar macrophages through GSK3 activity modulation

Reviewer #1 (Public review):

The manuscript by Rios et al. investigates the potential of GSK3 inhibition to reprogram human macrophages, exploring its therapeutic implications in conditions like severe COVID-19. The authors present convincing evidence that GSK3 inhibition shifts macrophage phenotypes from pro-inflammatory to anti-inflammatory states, thus highlighting the GSK3-MAFB axis as a potential therapeutic target. Using both GM-CSF- and M-CSF-dependent monocyte-derived macrophages as model systems, the study provides extensive transcriptional, phenotypic, and functional characterizations of these reprogrammed cells. The authors further extend their findings to human alveolar macrophages derived from patient samples, demonstrating the clinical relevance of GSK3 inhibition in macrophage biology.

The experimental design is sound, leveraging techniques such as RNA-seq, flow cytometry, and bioenergetic profiling to generate a comprehensive dataset. The study's integration of multiple model systems and human samples strengthens its impact and relevance. The findings not only offer insights into macrophage plasticity but also propose novel therapeutic strategies for macrophage reprogramming in inflammatory diseases.

Strengths:

(1) Robust Experimental Design: The use of both in vitro and ex vivo models adds depth to the findings, making the conclusions applicable to both experimental and clinical settings.
(2) Thorough Data Analysis: The extensive use of RNA-seq and gene set enrichment analysis (GSEA) provides a clear transcriptional signature of the reprogrammed macrophages.
(3) Relevance to Severe COVID-19: The study's focus on macrophage reprogramming in the context of severe COVID-19 adds clinical significance, especially given the relevance of macrophage-driven inflammation in this disease.

Weaknesses:

There are no significant weaknesses in the study.

Reviewer #2 (Public review):

Summary:

The study by Rios and colleagues provides the scientific community with a compelling exploration of macrophage plasticity and its potential as a therapeutic target. By focusing on the GSK3-MAFB axis, the authors present a strong case for macrophage reprogramming as a strategy to combat inflammatory and fibrotic diseases, including severe COVID-19. Using a robust and comprehensive methodology, in this study it is conducted a broad transcriptomic and functional analyses and offers valuable mechanistic insights while highlighting its clinical relevance

Strengths:

Well performed and analyzed

Weaknesses:

Additional analyses, including mechanistic studies, would increase the value of the study.

Author response:

Regarding a future revised version, we plan to:

• refer to the "MoMac-VERSE" study according to the original report.

• modify incorrectly formatted references.

• modify the text to acknowledge the heterogeneity and variability in the response of primary cells to the GSK3 inhibitor.

• improve the explanation of the reanalysis of single cell RNAseq data in Figure 7 (ref. 47, GSE120833), and re-adapt the graphs of the scRNA-Seq data using different plot parameters (e.g., reduction = "umap.scvi") to provide a more friendly-user visualization including bona fide macrophage markers for each subpopulation.

• include statistical analyses in each one of the figure legends

• perform additional analyses (e.g., dose-response and kinetics of CHIR-99021 effects) and mechanistic studies (e.g., role of proteasome) to further dissect the re-programming ability of the GSK3/MAFB axis.