Crocin Attenuates NLRP3 Inflammasome Activation by Inhibiting Mitochondrial Reactive Oxygen Species and Ameliorates Monosodium Urate-Induced Mouse Peritonitis

Round 1

Reviewer 1 Report

In this study, Sangare et al. investigated the inhibitory effect of Crocin on NLRP3 inflammasomes in mouse macrophages (both primary and cell line) and a mouse model of MSU-mediated peritonitis. The NLRP3 inflammasome is indeed a key player in initiating the inflammatory response in various disorders. One of the main advantages of this manuscript is the focus on Signal 2 of NLRP3 inflammasome activation with the use of several activators for this stage. Overall, the manuscript represents a great contribution to the field, and the experimental design well as data analysis are presented well.

Minor comments:

1.       In the Abstract, some abbreviations (e.g., AIM2 or ASC) are not introduced on the first usage. The authors also need to make sure that all full names are provided for the abbreviations. For example, the full name for ATP (adenosine triphosphate) is missing at the first mention (line 15).

2.       In the Abstract, it would be better to clarify that J774A.1 macrophage cells are murine. Otherwise, the origin is not clear.

3.       The data representation needs improvement (revision).  All figure bar graphs need to have a clear and concise group indication. It is possible to guess that numbers 125, 250, or 500 mean Crocyn dosage, and M refers to MCC950. Does the first bar in each figure represent untreated control? Could the authors make the labels for the bars easier to understand?

4.       Each presented Western blot image needs to have molecular weight labels showing protein sizes, in kDa.

5.       The sample size n should be provided for each figure. It might be better to indicate that data are expressed as the mean and standard error of the mean (SEM) not only in the Materials and Methods but also in figure legends.

6.       The authors may need to add the following recent paper examining the inhibiting effects of Crocin on NLRP3 inflammasome activation and mitochondrial superoxide production to their Discussion:

-          Zhang L, Jing M, Liu Q. Crocin alleviates the inflammation and oxidative stress responses associated with diabetic nephropathy in rats via NLRP3 inflammasomes. Life Sci. 2021. 278:119542. doi: 10.1016/j.lfs.2021.119542.

7.       Although the authors raised important questions about the importance of their study, the manuscript says nothing about the study limitations.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Crocin attenuates NLRP3 inflammasome activation by inhibiting mitochondrial reactive oxygen species and ameliorates 3 monosodium urate-induced mouse peritonitis.

Summary:  This manuscript describes the use of Crocin to modulate NLRP3 activation

Introduction: 

No comment

Materials and Methods:

2.4  This section needs more clarity.  Line 114-116. It is unclear whether these treatments are performed sequentially or are a part of the same procedure.  The authors fail to reference the origin of the chosen inflammasome trigger concentrations.

2.5  This section is not described in sufficient detail.  what is the experimental set-up? Need some reference to section 2.4.

2.6  It is unclear what was precipitated.  This would lead the reader to believe a solid is extracted from the supernatant.  Line 129 – pellet of what?

2.7  This section needs more detail.  Whole cell extracts or cytosolic, line 133?  Secondary antibodies not listed in section 2.1

2.8  What cell supernatants, pert. macrophage/ cell line/ both?

2.9    This section needs more detail or a reference to section 2.4.

2.10-2.12.  Authors need to indicate which cells in these assay or reference 2.4.

 2.14.  This manuscript would be improve using biological and technical replicate language. Line 185-86.  The authors failed to demonstrate that all the data represents normal distribution and therefore cannot justify the use of  a parametric test as the first part of the analysis.

Results

Overview- the authors fail to provide the reader with specific details associated with the data.  For example, using vague language such as “increases” or “decrease”.  The manuscript would be improved by conveying the exact changes and standard errors since these values cannot be determined from the graphs and other data.

Figure 1A-  The figure’s xaxis contain tick marks for the first two conditions, making it difficult to interpret.  This should be corrected in all figures.  In addition, the format of the bars and the abbreviations of Nig, ATP, MSU below are confusing, and readers will assume the concentrations listed corresponded to those labels. 

The authors failed to use MCC950 or other positive controls for the other experimental groups.

Figure 2B- The WB image for pro caspase 1 is overexposed, making it difficult to determine qualitative differences between treatments. If the levels of casp-1 p20 are reduced, the reverse should be seen for the Pro-casp 1 form.  However, this trend is not seen, regardless of inflammasome trigger or concentration of Crocin applied.

Figure 2- description should mention the same incubation periods were used, as described in figure 1.  Referring figure 1 in another figure legend is insufficient.

Figure 3- Supernatant was use for WBlotting, yet this sample processing was never described in MM.

 

The macrophages are extracted from mice, but no indication regarding if the cells were immediately used for experimentation or briefly cultivated.  This process will induce cell death from a percent of the cells, yet the cells show no IL1 secretion. 

Figure 4A-  The WBlott bands for proIL-beta are degraded and a different blot needs to be provided that is more clear.

Figure 5 – Line 264-265.  This sentence is unclear “out of cells”.  The author use fluorescent microscopy to image ASC speck.  The authors fail to describe  that the speck represents ASC concentration, identified by the ASC antibody.   The authors fail to provide a negative control represented by secondary antibody alone treatment.  The authors fail to provide the method in which the specks were tabulated…manually? ImageJ?

Figure 7 – The authors failed indicate if sham animal data is represented. The flow cytometry data shows a moderate shift from the upper right quadrant after Crocin pretreatment, however the authors fail to provide the quantitative data to assert the shift was significant.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

Brief Summary:

 

This study investigated the inhibitory effect of crocin (hydrophilic carotenoid pigment) on the NLRP3 inflammasome. Crocin was found to reduce the activation of NLRP3 inflammasome and inhibit the secretion of IL-1β, activation of caspase-1 and GSDMD in response to the NLRP3 inflammasome triggers such as Nigericin, ATP, and MSU. The results showed that crocin acts as a NLRP3 inflammasome-specific inhibitor and suggests that crocin might relieve NLRP3 inflammasome-related diseases. Crocin was also observed to reduce ASC oligomerization, mtROS production, and mitochondrial damage induced by NLRP3 inflammasome triggers. The authors showed that crocin can inhibit not only the secretion of IL-1b and IL18 but also the recruitment of neutrophils in MSU-induced peritonitis model. These results suggest that crocin might have therapeutic potential for diseases related to the NLRP3 inflammasome, including rheumatoid arthritis, systemic lupus erythematosus, and gout.

 

Major Revision:

 

1. Authors needs to assess the effect of Crocin alone on cell viability in a dose dependent manner. Even though Fig. 1C shows that Crocin ameliorated the decreased cell viability following Nig, ATP and MSU treatments, one needs to assess the toxicity dosage of Crocin on cells.  

 

2. In Fig 4D, it can be clearly observed that the MitoTracker deep red either stays the same or even increases following the MSU treatment. MitoTracker Green also increases following the MSU treatment and then decreases with Crocin treatment. Results shown in Fig 4C and 4D does not validate authors’ conclusions in lines 277 – 282 “As expected, MSU stimulation……..”. This needs to be addressed before the authors can conclude that Crocin suppresses mitochondrial damage thereby decreasing NLRP3 inflammasome activation.

 

3. In respect to figure 1B, authors need to address whether the active p20 Caspase-1 and p17 IL-1b is in the cytosolic extract, as Crocin also inhibits GSDMD cleavage and pyroptosis as shown in section 3.2. This can be addressed by performing a western blot for p20 Caspase-1 and p17 IL-1b in the cytosolic extract (lysate fraction) using the same conditions as outlined in Fig 1B.

 

4. In Fig 3B, authors need to also assess the GSDMD and pro-GSDMD proteins in the cytosolic extract (lysate) by western blot. 

 

Minor Revision:

 

1. Line 71: Please revise the sentence in the Introduction section line 71 "These plants are primarily used….." as it is the same sentence in the abstract line 10. Using the same sentence in the abstract and the Introduction section can be considered as self-plagiarism.

 

2. Line 74: Typo "," instead of "." after [11,12]

 

3. Line 10, 71, 277, 278, 279, 280 : Change red font to black

 

4. Line 156: letter "m" is missing in the sentence "resuspend in 500  l PBS,"

 

5. Fig 1A, 2B-C, 3A-C, 4B-C, 5C, 6A, 6C, 7A-D: Please also include “Cro (uM)” accompanying the crocin concentration 0, 125,250 and 500 uM.

 

6. Fig 2B: Typo "Nlig" instead of "Nig"

 

7. Fig 6D: Typo “Mitoracker” instead of “Mitotracker”

 

8. In Figure 5B, it can be observed that the nucleus is visibly smaller following the Nigericin treatment. Authors needs to make this distinction in the results section 3.5 and add a reference if this phenomenon was previously observed in other studies.  

 

9. In line 323, authors include pro-IL-1b as one of the components of NLRP3 inflammasome. Pro-IL-1b is not a part of NLRP3 inflammasome. This needs to be addressed.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

Thank you for the updated manuscript. 

For Major Revision point 3, Authors replied that "I added the WB images for active p20 Caspase-1 and p17 IL-1b in the cytosolic extract (lysate fraction) in figure 1 and 3." 

However, the western blot of p17 IL-1b in the cytosolic extract (lysate) was not added in figure 1 and 3. Please add these WB images.

 

Please remove “p42” after Actin in all the relevant figures (1B, 2A,3B,4A,5A).

 

Author Response

Thank you for reviewing my manuscript.

We revised the manuscript (figures) according to reviewer’s comments and appreciate for your help to improve my manuscript.

The response to reviewer’s comments are listed below.

 

For Major Revision point 3, Authors replied that "I added the WB images for active p20 Caspase-1 and p17 IL-1b in the cytosolic extract (lysate fraction) in figure 1 and 3."

However, the western blot of p17 IL-1b in the cytosolic extract (lysate) was not added in figure 1 and 3. Please add these WB images.

→ I am sorry I made a mistake when I wrote the response to reviewer’s comment; I didn’t add the image for p17 IL-1b. Unfortunately, we could not detect p17 IL-1b in the cytosolic extract (lysate), so I couldn’t add. However, we showed that Crocin inhibited the level of p17 IL-1b in cell supernatant (that is, secretory p17 IL-1b) using Western blotting and ELISA. The level of p17 IL-1b in the cytosolic extract is not thought to be critical.

 

Please remove “p42” after Actin in all the relevant figures (1B, 2A,3B,4A,5A).

→ I removed “p42” after Actin in figures (1B, 2A, 3B, 4A, 5A).