HSP90 Inhibition Disrupts 27-Hydroxycholesterol-Induced Inflammatory Signaling in Monocytic Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors present a study in which they examine the effects of HSP90 in 27-hydroxycholesterol-induced inflammatory signaling. I have several minor comments:
1. The image quality is too blurry. Please provide a higher resolution version.
2. Does flow cytometry have a control of the same type? This is of great significance for the streaming analysis of gate patterns.
3.How specific is ganetespib? Will it also inhibit other proteins?
4.The text part of the picture has appeared with wavy lines. Please make the correction.

Author Response

Reviewer 1.

1.The image quality is too blurry. Please provide a higher resolution version.

We appreciate the reviewer’s comment. The images have been replaced with higher-resolution versions throughout the manuscript.

2. 
   Does flow cytometry have a control of the same type? This is of great significance for the streaming analysis of gate patterns.

We appreciate the reviewer’s comment. In each experiment, we included a negative control in which the cells were processed without staining, and this served as a reference for gating pattern analysis.

3.How specific is ganetespib? Will it also inhibit other proteins?

We thank for the reviewer’s insightful comment. Ganetespib is a non-ansamycin inhibitor that binds to the N-terminal ATP-binding pocket of HSP90, with high potency against the cytosolic isoforms HSP90α and HSP90β. Through this mechanism, it indirectly destabilizes a broad spectrum of HSP90 “client” proteins, including HER2/ERBB2, EGFR, AKT, and JAK2, leading to their degradation. [PMID:29717218]

In addition, kinome-wide profiling studies have shown that, at higher concentrations (e.g., 10 µM in biochemical screens), ganetespib can directly inhibit several kinases such as ABL1/ABL2, DDR1, and TRKA-TFG, in some cases with sub-micromolar IC₅₀ values. This indicates that while its primary and most relevant target is HSP90, off-target kinase inhibition may occur under certain conditions. [PMID:34077750]

However, in our study the maximal concentration of ganetespib used was 1.3 µM (0.5µg/ml), a range in which the compound is widely considered to act primarily through HSP90 inhibition. Thus, it is reasonable to conclude that the observed effects are attributable to HSP90 blockade and client protein destabilization rather than to direct off-target kinase inhibition.

4.The text part of the picture has appeared with wavy lines. Please make the correction.

We appreciate the reviewer’s careful comment, and the figure has been corrected as requested.

Reviewer 2 Report

Comments and Suggestions for Authors

In the present study the authors studied the effect of HSP90 inhibition on 27-hydroxycholesterol-induced in-2 
inflammatory signaling in monocytic cells.

The results are interesting but more data is needed.

1. The authors need to estimate IL-6 and TNF in the supernatants of cultures monocytic cells in the presence of 27-hydroxycholesterol and HSP protein or stimulants.
2. In animals, the authors can admin r27-hydroxycholesterol and study plasma and tissue levels of IL-6 and TNF (tissue may include liver).
3. Is there any role for other HSPs also?

  

  

Comments on the Quality of English Language

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Author Response

Reviewer 2.

1. The authors need to estimate IL-6 and TNF in the supernatants of cultures monocytic cells in the presence of 27-hydroxycholesterol and HSP protein or stimulants.

We thank the reviewer for the insightful suggestion. In line with the reviewer’s request, we examined the expression of TNF-α and assessed whether ganetespib could suppress it. Our results showed that 27-hydroxycholesterol significantly induced TNF-α expression, both at the cellular level and in the culture supernatant (p < 0.001). As expected, ganetespib markedly reduced this 27-hydroxycholesterol-induced TNF-α expression (p < 0.001). By contrast, under our current experimental conditions, 27-hydroxycholesterol did not induce IL-6 expression. These results are presented in Supplementary Figure 4.

 

2. In animals, the authors can admin 27-hydroxycholesterol and study plasma and tissue levels of IL-6 and TNF (tissue may include liver).

We appreciate the reviewer’s valuable suggestion. In our previous attempts, we administered 27-hydroxycholesterol intraperitoneally in a mouse model to investigate inflammatory cytokine expression and M1 polarization of peritoneal macrophages. We, however, were not able to obtain the data demonstrating that 27-hydroxycholesterol increased the inflammatory response in resident mouse peritoneal macrophages. In addition, treatment of the murine macrophage cell line RAW264.7 with 27-hydroxycholesterol also did not induce inflammatory responses.

Although the exact reason for this unresponsiveness remains unclear, these findings suggest that 27-hydroxycholesterol elicits inflammatory response more prominently in human monocytic cells rather than in murine macrophages. This interpretation is consistent with previous reports that have documented functional differences between human and mouse macrophages, including differences in lipid sensing and inflammatory signaling pathways. [PMID:33281821] Taken together, our data and these reports indicate that the pro-inflammatory response of 27-hydroxycholesterol may be species-dependent.

 

3. Is there any role for other HSPs also?

We appreciate the reviewer’s insightful question. We carefully searched the literature for evidence that ganetespib directly inhibits heat shock proteins other than HSP90. To our knowledge, there is no peer-reviewed study demonstrating direct inhibition of HSP70, HSP27, HSP40, or HSP105 by ganetespib. Most primary data and clinical reports identify cytosolic HSP90α/β as the principal molecular targets of ganetespib. Accordingly, our interpretation focuses on HSP90 inhibition rather than direct suppression of other heat shock proteins.