Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Authors investigated potential role of histones usually confined to nucleosomes released into the bloodstream during sepsis due to cell damage. They hypothesized that released histones contribute to tributing to organ dysfunction, specifically sepsis-associated encephalopathy (SAE). Investigators used a cecal ligation and puncture (CLP)-induced polymicrobial sepsis model and evaluated histone release, blood-brain barrier (BBB) disruption, complement activation, and glial responses in the brain. Immunostaining revealed histone accumulation and increased soluble histone levels in the brain 8 - 24 hours post-CLP. BBB permeability increased, confirmed by FITC-inulin and Texas Red-dextran clearance assays. There was cdomplement activation (detected by PCR), along with increased levels of activation astrocyte markers (GFAP, S100),  GFAP-positive astrocytes and Iba1-positive microglia occurred post-CLP.  Histones were detected in astrocytes and microglia. Exposure of astrocytes in vitro released histones upon activation and also demonstrated the ability to uptake extracellular FITC-labeled histones. Histone exposure elevated intracellular calcium levels and triggered cytokine secretion in astrocytes. Histone stimulation activated the NLRP3 inflammasome, amplifying inflammation. These findings suggest that histone release during sepsis drives neuroinflammation, BBB disruption, and glial activation. Study is very infesting and novel as role of histones in sepsis has not been studied before. Few manor issues require further clarification.

1. Authors clearly demonstrated BBB compromise in functional assays. It would be good also to show changes of tight junction proteins by immunostaining.
2. While study concentrated on astocytes, what might be role of microglia in histone release?
3. It is not unclear what is justification for use of LPS plus ATP in astrocyte

Author Response

Response to the Reviewers’ Comments/Critiques:

 

We thank the Editor and the Reviewers for their careful evaluation of our manuscript and for their constructive and insightful comments. We have addressed each comment point by point below. All revisions have been made in the manuscript, with changes highlighted in yellow to facilitate review.

 

1: “Authors clearly demonstrated BBB compromise in functional assays. It would be good also to show changes of tight junction proteins by immunostaining.”

Response: We agree that assessment of tight junction proteins by immunostaining would provide complementary structural insight alongside our functional BBB permeability assays. In the current study, we focused on quantitative, size-selective functional measurements (FITC-inulin and Texas Red-dextran clearance), which directly assess BBB integrity in vivo and are highly sensitive to early barrier disruption. Future studies will include immunostaining and biochemical analysis of endothelial tight junction proteins (e.g., claudin-5, occludin, ZO-1) to further delineate the structural mechanisms underlying histone- and complement-mediated BBB injury.

This point has been addressed in the Discussion in the context of our BBB integrity data (Lines 434-438).

 

2: “While study concentrated on astrocytes, what might be role of microglia in histone release?”

Response: We agree with the Reviewer that microglia may represent an additional cellular source influencing histone release and downstream neuroinflammatory signaling during sepsis. This important aspect will be addressed in future investigations. In the present study, we focused on astrocytes because of their strategic localization at the neurovascular unit, their known responsiveness to complement activation, and their capacity to directly influence BBB integrity. Our immunofluorescence data demonstrate histone accumulation in both astrocytes and microglia in vivo, suggesting that microglia may also participate in histone uptake and/or release. Future studies will specifically determine whether microglia actively release histones and define their downstream signaling responses in comparison to astrocytes.

We added clarification on this point in the Discussion (Lines: 441-453).

 

3: “It is not unclear what is justification for use of LPS plus ATP in astrocyte.”

Response: We apologize for the lack of clarity regarding the justification for using LPS plus ATP in astrocytes. LPS priming followed by ATP stimulation is a well-established experimental paradigm used to activate the NLRP3 inflammasome in many cell types, including phagocytes and other innate immune cells (PMID: 36127465). Here, we used the same approach in astrocytes to determine whether they possess the molecular machinery required for inflammasome activation in response to inflammatory stimuli, consistent with prior studies (references cited in the revised manuscript).

We have clarified this justification in the revised manuscript (Results Section 2.7; Lines 320-327).

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, the authors examine the role of extracellular histones in sepsis-associated encephalopathy using a CLP-induced polymicrobial sepsis model. They report increased histone accumulation in the brain, blood–brain barrier disruption, complement activation, and activation of astrocytes and microglia following sepsis. Histones were detected within both glial cell types, and in vitro experiments demonstrate that astrocytes can release and uptake extracellular histones, leading to calcium signaling, cytokine release, and NLRP3 inflammasome activation. Overall, the study suggests that extracellular histones contribute to neuroinflammation and BBB dysfunction during sepsis. The study is carried out well, and is of importance to the field. Here are some of my minor suggestions:

 

1. Section 2.4: The sentence stating, “Notably, a recent study observed astrocyte activation (increased GFAP expression) in frontal brain biopsies from three humans who died of sepsis, compared to brain tissue from five non-septic individuals [39],” appears to be more appropriate for the Discussion section rather than the Results, as it refers to previously published findings rather than data generated in the current study.
2. Section 2.5: It is interesting that the authors report histone localization in both microglia and astrocytes in CLP-induced septic mice. It would further strengthen this observation if the authors quantified the degree of colocalization using Pearson’s correlation coefficient (e.g., in ImageJ) across all images. This analysis could reveal whether histone colocalization differs between astrocytes and microglia, or if one cell type shows significantly higher association than the other.
3. Section 2.5: The authors use the BWA3 antibody to demonstrate histone immunofluorescence. A brief description of this antibody would be helpful—for example, specifying that BWA3 recognizes an epitope common to histones H2A and H4.
4. Did the authors investigate whether microglia are also capable of taking up extracellular histones, similar to astrocytes? Additionally, downstream analyses such as calcium release, NLRP3 inflammasome activation, and subsequent functional assays appear to have been performed only in astrocytes. If these experiments were not conducted in microglia, it would be helpful for the authors to address this choice in the Discussion and explain why astrocytes were prioritized over microglia for these analyses.
5. I am somewhat confused by the sequence of events presented in the study. According to the schematic in Figure 8, complement activation is depicted as one of the earliest events. However, the data indicate that complement upregulation and activation occur at 8 hours, and increased BBB permeability is also evident starting at 8 hours. In contrast, histone uptake is observed as early as 4 hours. Clarification of this would be helpful.

 

Author Response

Response to the Reviewers’ Comments/Critiques:

 

We thank the Editor and the Reviewers for their careful evaluation of our manuscript and for their constructive and insightful comments. We have addressed each comment point by point below. All revisions have been made in the manuscript, with changes highlighted in yellow to facilitate review.

 

1: “Section 2.4: The sentence stating, “Notably, a recent study observed astrocyte activation (increased GFAP expression) in frontal brain biopsies from three humans who died of sepsis, compared to brain tissue from five non-septic individuals [39],”

appears to be more appropriate for the Discussion section rather than the Results, as it refers to previously published findings rather than data generated in the current study.”

Response: We appreciate this comment. The sentence has been moved from Results Section 2.4 to the Discussion section, as suggested. In addition, we consolidated overlapping text describing human astrocyte activation from Warford et al. (PMID 27832827) into a single interpretive statement in the Discussion, retaining the reported human sample details while avoiding redundancy (Lines 454-459).

 

2: “Section 2.5: It is interesting that the authors report histone localization in both microglia and astrocytes in CLP-induced septic mice. It would further strengthen this observation if the authors quantified the degree of colocalization using Pearson’s correlation coefficient (e.g., in ImageJ) across all images. This analysis could reveal whether histone colocalization differs between astrocytes and microglia, or if one cell type shows significantly higher association than the other.”

Response: We acknowledge that quantitative assessment of signal overlap would provide a more rigorous evaluation of histone localization within glial populations. In the present study, immunofluorescence was used primarily to establish spatial association of histones with astrocytes and microglia at a qualitative level. Future work will incorporate established quantitative colocalization approaches (e.g., Pearson’s or Manders’ coefficients) to enable direct, cell-type-specific comparisons of histone association across glial subtypes.

We have added a statement in the Discussion to acknowledge this limitation and outline planned future analyses (Lines: 445-448).

 

3: “Section 2.5: The authors use the BWA3 antibody to demonstrate histone immunofluorescence. A brief description of this antibody would be helpful—for example, specifying that BWA3 recognizes an epitope common to histones H2A and H4.”

Response: We appreciate this helpful comment. The BWA3 monoclonal antibody recognizes a shared epitope present on core histones H2A and H4, which exhibit conserved sequence and structural homology. As previously described for anti-histone monoclonal antibodies derived from autoimmune mice (Monestier et al., 1993, PMID: 8366857), this antibody does not distinguish between individual histone subtypes but instead detects histone exposure based on a common epitope shared by H2A and H4.

As suggested by the Reviewer, we have added a description of the BWA3 antibody, along with the appropriate reference, to the Methods section (Lines: 566-568):

 

4: “Did the authors investigate whether microglia are also capable of taking up extracellular histones, similar to astrocytes? Additionally, downstream analyses such as calcium release, NLRP3 inflammasome activation, and subsequent functional assays appear to have been performed only in astrocytes. If these experiments were not conducted in microglia, it would be helpful for the authors to address this choice in the Discussion and explain why astrocytes were prioritized over microglia for these analyses.”

Response: We appreciate this reviewer comment and agree that addressing potential microglial histone uptake and the rationale for prioritizing astrocytes in downstream analyses is important. We did not perform functional assays assessing microglial uptake of extracellular histones or downstream responses such as calcium signaling or NLRP3 inflammasome activation in this study. However, our in vivo immunofluorescence data demonstrate histone localization within microglia, suggesting their potential involvement. We prioritized astrocytes for mechanistic and functional analyses because of their abundance, direct interaction with the BBB, and well-established role in regulating barrier integrity and neurovascular inflammation. In addition, rigorous functional interrogation of microglia would require additional isolation strategies and cell-type-specific assays to ensure purity and physiological relevance, which were beyond the scope of the present study. These analyses will be the focus of future investigations.

We have added text to the Discussion explicitly outlining this rationale (Lines 448-453), as also noted in our response to R1.2.

 

5: “I am somewhat confused by the sequence of events presented in the study. According to the schematic in Figure 8, complement activation is depicted as one of the earliest events. However, the data indicate that complement upregulation and activation occur at 8 hours, and increased BBB permeability is also evident starting at 8 hours. In contrast, histone uptake is observed as early as4 hours. Clarification of this would be helpful.”

Response: We appreciate the reviewer’s comment regarding the apparent sequence of events. The schematic in Figure 8 is intended to depict interacting and overlapping processes rather than a strictly linear temporal sequence. Our data indicate that histone accumulation in the brain can be detected as early as 4 hr post-CLP, whereas measurable BBB permeability changes and complement activation become evident around 8 hr. We propose that these processes occur in parallel and synergistically contribute to BBB dysfunction and glial activation.

Accordingly, we revised the Discussion (Line 367, Lines: 418-423) and the Figure 8 legend to emphasize overlapping and interacting processes rather than a strictly linear sequence.