Tolerance to Lung Infection in TWIK2 K⁺ Efflux Mediated Macrophage Trained Immunity

Reviewer #1 (Public review):

Summary:

Alveolar macrophages (AMs) are key sentinel cells in the lungs, representing the first line of defense against infections. There is growing interest within the scientific community in the metabolic and epigenetic reprogramming of innate immune cells following an initial stress, which alters their response upon exposure to a heterologous challenge. In this study, the authors show that exposure to extracellular ATP can shape AM functions by activating the P2X7 receptor. This activation triggers the relocation of the potassium channel TWIK2 to the cell surface, placing macrophages in a heightened state of responsiveness. This leads to the activation of the NLRP3 inflammasome and, upon bacterial internalization, to the translocation of TWIK2 to the phagosomal membrane, enhancing bacterial killing through pH modulation. Through these findings, the authors propose a mechanism by which ATP acts as a danger signal to boost the antimicrobial capacity of AMs.

Strengths:

This is a fundamental study in a field of great interest to the scientific community. A growing body of evidence has highlighted the importance of metabolic and epigenetic reprogramming in innate immune cells, which can have long-term effects on their responses to various inflammatory contexts. Exploring the role of ATP in this process represents an important and timely question in basic research. The study combines both in vitro and in vivo investigations and proposes a mechanistic hypothesis to explain the observed phenotype.

Weaknesses:

Although these findings are convincing and intrinsically interesting, they do not support the conclusion that ATP induces trained immunity. By definition, trained immunity refers to long-lasting metabolic and epigenetic reprogramming initiated by a primary stimulus. Importantly, some of these changes persist after the cells have returned to a basal activation state, thereby generating an altered response upon secondary stimulation (https://doi.org/10.1038/s41590-020-00845-6). In the present study, the data demonstrate a sustained increase in inflammasome activation and enhanced microbicidal activity for up to seven days following ATP exposure. While this sustained activation is noteworthy as well as metabolic shift, it does not demonstrate the existence of trained immunity. The terms priming or sustained activation would therefore be more appropriate than trained immunity.

Similarly, the observation of increased chromatin accessibility at inflammasome-related genes is expected given the robust activation of this pathway. The presence of open chromatin at these loci does not, by itself, constitute evidence for long-term trained immunity. The authors should therefore be cautious with their terminology and avoid overinterpreting their findings.

The authors have revised the manuscript to address the comments raised during the first rounds of review. However, several figures, figure legends, and methodological sections still require additional adjustments and clarification.

The Methods section remains incomplete and requires substantial revision. For instance, the methodology used to quantify immune cell populations presented in Figure 2 is still not described. It is not stated how immune cells were isolated and identified (e.g. flow cytometry from lung tissue). No information is provided regarding tissue digestion, cell isolation procedures, or gating strategy (presumably by flow cytometry). These details are essential and should be included, together with the corresponding gating strategy and absolute cell numbers.

There are inconsistencies throughout the manuscript. For example, the authors report n = 3 in the figure legend 2 and 3 independent experiments, whereas 3 or 4 points are represented in the graphs. This discrepancy is unclear and should be clarified.

Overall, while the study addresses an interesting biological question, the manuscript would benefit from substantial revision prior to publication. In particular, clarifications and improvements regarding the methodology, data presentation, and interpretation are required to strengthen the rigor and reproducibility of the conclusions. Several of the conclusions extend beyond what is directly supported by the data. In particular, the interpretation that these findings demonstrate trained immunity should be revised, and additional methodological clarifications and corrections are required.

Author response:

The following is the authors’ response to the previous reviews

Public Reviews:

Reviewer #1 (Public review):

Please include an uptake control (early time point) or time‑course to distinguish phagocytosis from intracellular killing.

We agree that distinguishing uptake from intracellular killing is important for interpreting bactericidal assays. Due to a recent transition, we are unable to conduct additional early‑time‑point assays. To address this transparently, we have revised the manuscript to clarify that our measurements represent overall bacterial load reduction, reflecting the combined effects of uptake and killing.

The normalization as ‘fold killing’ is non‑standard; please report absolute CFU (log scale).

We have retrieved the raw data and re‑expressed all bactericidal activity measurements as absolute CFU. All relevant figures, legends, and text have been updated accordingly.

Authors report quantification of cytokine concentrations, yet no information is provided regarding how these measurements were performed.

Cytokine concentrations were quantified by ELISA. We have now added details regarding assay kits, sample preparation, and detection parameters to the Methods section.

While the choice of IL‑1β and IL‑6 is straightforward, the focus on IL‑18 requires explicit justification.

IL‑18 is a macrophage‑associated pro‑inflammatory cytokine with established links to inflammasome activation and trained immunity pathways, providing a clear justification for its inclusion.

The methodology used to quantify immune cell populations presented in Figure 2 is not described.

We have added a detailed description of the flow cytometry methodology, including staining and gating strategies.

Immune cell quantification would be expected in the context of the challenge experiment as well.

While we agree that such data would be valuable, additional mouse experiments cannot be performed because the animals used in the challenge model are not currently available during the transition period. If feasible, we are exploring ex vivo flow cytometry data from TWIK2 mutant versus wild‑type macrophages.

AMs are not considered recruited immune cells; this should be corrected.

We have corrected this in the figure legend and throughout the manuscript.

The authors report n = 5 for the survival curves in the figure legend, whereas n = 7 is stated in the Methods section.

We have corrected the sample size to ensure consistency between the figure legend and Methods.

ATAC‑seq peaks are referred to as ‘genes’ and ‘differentially expressed genes’.

We have corrected the terminology in the manuscript. ATAC‑seq identifies differentially accessible chromatin regions, which are then annotated to the nearest downstream gene.

In Figure 7, trained WT and Nlrp3-/- mice display similar levels of bacterial clearance. How should this result be interpreted?

A portion of this phenotype is via the normalization of phagocytosis to ‘fold killing’. Presentation of the raw CFU data shows a trend towards reduced bacterial clearance in Nlrp3-/- mice.

Reviewer #2 (Public review):

Sample numbers for experiments 1, 2, and 6 are not provided.

We have added explicit n values for all experiments and verified their accuracy against the original records.

The Discussion would benefit from a clear summary of study caveats.

We agree and have added a dedicated paragraph outlining key Caveats as described.

Specific identities of DEGs are not provided; only pathway enrichment is shown.

We have now included a supplementary table listing the differentially expressed genes identified in our analysis.

Controls for subcellular fractionation and dye microscopy should be included.

Controls for subcellular fractionation have added in figure 3B and controls for dye microscopy have now been added in supplementary figure 1.

The text states that protease inhibitors diminish ATP‑induced training effects, but the figure does not show significance.

We have re‑examined the data and updated the figure to include statistical testing where appropriate.