Microbiota from young mice counteracts susceptibility to age-related gout through modulating butyric acid levels in aged mice

Reviewer #1 (Public review):

Summary:

In their manuscript the authors report that fecal transplantation from young mice into old mice alleviates susceptibility to gout. The gut microbiota in young mice is found to inhibit activation of the NLRP3 inflammasome pathway and reduce uric acid levels in the blood in the gout model.

Strengths:

They focused on the butanoate metabolism pathway based on the results of metabolomics analysis after fecal transplantation and identified butyrate as the key factor in mitigating gout susceptibility. In general, this is a well-performed study.

Weaknesses:

The discussion on the current results and previous studies regarding the effect of butyrate on gout symptoms is insufficient. The authors need to provide a more thorough discussion of other possible mechanisms and relevant literature.

Reviewer #2 (Public review):

Summary:

The revised manuscript presents interesting findings on the role of gut microbiota in gout, focusing on the interplay between age-related changes, inflammation, and microbiota-derived metabolites, particularly butyrate. The study provides valuable insights into the therapeutic potential of microbiota interventions and metabolites for managing hyperuricemia and gout. While the authors have addressed many of the previous concerns, a few areas still require clarification and improvements to strengthen the manuscript's clarity and overall impact.

(1) While the authors mention that outliers in the data do not affect the conclusions, there remains a concern about the reliability of some figures (e.g., Figure 2D-G). It is recommended to provide a more detailed explanation of the statistical analysis used to handle outliers. Additionally, the clarity of the Western blot images, particularly IL-1β in Figure 3C, should be improved to ensure clear and supportive evidence for the conclusions.
(2) The manuscript raises a key question about why butyrate supplementation and FMT have different effects on uric acid metabolism and excretion. While the authors have addressed this by highlighting the involvement of multiple bacterial genera, it is still recommended to expand on the differences between these interventions in the discussion, providing more mechanistic insights based on available literature.
(3) It is noted that IL-6 and TNF-α results in foot tissue were requested and have been added to supplementary material. However, the main text should clearly reference these additions, and the supplementary figures should be thoroughly reviewed for consistency with the main findings. The use of abbreviations (e.g., ns for no significant difference) and labeling should also be carefully checked across all figures.
(4) The manuscript presents butyrate as a key molecule in gout therapy, yet there are lingering concerns about its central role, especially given that other short-chain fatty acids (e.g., acetic and propionic acids) also follow similar trends. The authors should consider further acknowledging these other SCFAs and discussing their potential contribution to gout management. Additionally, the rationale for focusing primarily on butyrate in subsequent research should be made clearer.
(5) The full-length uncropped Western blot images should be provided as requested, to ensure transparency and reproducibility of the data.
(6) Despite the authors' revisions, several references still lack page numbers. Please ensure that all references are properly formatted, including complete page ranges.
The manuscript has improved with the revisions made, particularly regarding clarifications on experimental design and the inclusion of supplementary data. However, some concerns about data quality, mechanistic insights, and clarity in the figures remain. Addressing these points will enhance the overall impact of the work and its potential contribution to the understanding of the gut microbiome in gout and hyperuricemia. A final revision, with careful attention to both major and minor points, is highly recommended before resubmission.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #3 (Public Review):

Some critical comments are provided below：

(1) The data quality still needs to be improved. There are many outliers in the experimental data shown in some figures, e.g. Figure 2D-G. The presence of these outliers makes the results unreliable. The author should thoroughly review the data analysis in the manuscript. In addition, a couple of western blot bands, such as IL-1β in Figure 3C, are not clear enough, please provide clearer western blot results again to support the conclusion.

Following our comparative analysis, we have determined that these data do not affect our conclusions. Moreover, our experimental design included a total of six mice per group, with all mouse samples being subjected to testing.

(2) As shown in Figure 1G-I, foot thickness and IL-1β content in foot tissues of the Aged+Abx group were significantly reduced, but there was no difference in serum uric acid level. In addition, the Abx-untreated group should be included at all ages.

Thank you for your comment. We have included this data in Supplemental Material 4.

(3) Since FMT (Figure 4) and butyrate supplementation (Figure 8) have different effects on uric acid synthesis enzyme and excretion, different mechanisms may lie behind these two interventions. Transplantation with significantly enriched single strains from young mice, such as Bifidobacterium and Akkermansia, is the more reliable approach to reveal the underlying mechanism between gut microbiota and gout.

Thank you for your comment. Due to the involvement of multiple bacterial genera in gout and hyperuricemia, and the practical challenge of testing all strains, our focus shifted to the functional implications and metabolism of the microbiota. Experimental validation confirmed that butyrate exerts a dual-therapeutic effect in mitigating gout and hyperuricemia.

(4) In Figure 2F, the results showed the IL-1β, IL-6, and TNF-α content in serum, which was inconsistent with the authors' manuscript description (Line 171).

Thank you for your comment. The modifications to the results have been implemented.

(5) Figures 2F-H duplicate Supplementary Figures S1B-D. The authors should prepare the article more carefully to avoid such mistakes.

Thank you for your comment. We have corrected it in the manuscript.

(6) In lines 202-206, the authors stated that the elevated serum uric acid levels in the Young+Old or Young+Aged groups, but there is no difference in the results shown in Figure 4A.

Thank you for your comment. We have corrected it in the manuscript.

(7) Please visualize the results in Table 2 in a more intuitive manner.

The results have been presented in Table 2 with a more intuitive visual format. The detailed information is presented in Supplement 4.

(8) The heatmap in Figure 7A cannot strongly support the conclusion "the butyric acid content in the faeces of Young+PBS group was significantly higher than that in the Aged+PBS group". The author should re-represent the visual results and provide a reasonable explanation. In addition, please provide the ordinate unit of Supplementary Figure 7A-H.

Thank you for your comment. Figure 7A and Supplementary Figure 7A-H together illustrate "the butyric acid content in the faeces of Young+PBS group was significantly higher than that in the Aged+PBS group", and the specific units of short-chain fatty acids have been annotated in the manuscript.

(9) Uncropped original full-length western blot should be provided.

Thank you for your comment. We have made relevant notes in the paper.

Reviewer #1 (Recommendations For The Authors):

Gout, a prevalent form of arthritis among the elderly, exhibits an intricate relationship with age and gut microbiota. The authors found that gut microbiota plays a crucial role in determining susceptibility to age-related gout. They observed that age-related gut microbiota regulated the activation of the NLRP3 inflammasome pathway and modulated uric acid metabolism. "Younger" microbiota has a positive impact on the gut microbiota structure of old or aged mice, enhancing butanoate metabolism and butyric acid content. Finally, they found butyric acid exerts a dual effect, inhibiting inflammation in acute gout and reducing serum uric acid levels. This work's insights emphasize the potential of "young" gut microbiome in mitigating senile gout. The whole study was interesting, but there were some minor errors in the overall writing of the paper. The author should carefully check the spelling of the words in the text and the case consistency of the group names.

Questions:

(1) Line 118, line 142, and elsewhere 24 months in the same format as before.

Thank you for your comment. We have corrected it in the manuscript.

(2) Lines 123, Old and Aged group should be a complex number.

Thank you for your suggestion. We have corrected it in the manuscript.

(3) Why does line 133 mention the use of ABX? Please add a brief explanation.

Thank for your suggestion. The aim of utilizing ABX is to construct the linkage between gut microbiota, age, and gout.

(4) Lines 172-175, the description of TNF does not match the description of the result figure, may be the picture placement error, please correct this.

Thank you for your careful review. The error has been corrected and the accurate result has been inserted into the original manuscript.

(5) Lines183-185 and lines193-lines195, Pro-Caspase-1 and Pro-IL activate excess write.

Thank you for your careful review. We have corrected the error at the original location.

(6) Line 400, the text should not be written as increased.

Thank you for your careful review. We have corrected the error at the original location.

(7) "ns" needs to be added in the legend to indicate that there is no significant difference.

Thank you for your careful review. We have corrected the error at the original location.

(8) Lines 1080-1084 "Old or Aged control group and the old or aged group", group names should be case-sensitive.

Thank you for your suggestion. We have made the correct modification to the group names.

(9) Lines 1072-1073, "Representative western blot images of foot tissue NLRP3 pathways proteins" add band density.

Thank you for your suggestion. We have corrected the error on lines 1072-1073 of the article.

Reviewer #2 (Recommendations For The Authors):

Specific comments:

(1) In Figures 1G-H, the Aged+PBS group with antibiotic treatment shows a significant reduction in foot swelling and IL-1β compared to the Young+PBS and Old+PBS groups. The authors state that age-related changes in the gut microbiota exacerbate gout. However, why does only the Aged+PBS group improve with antibiotic treatment? It seems that butyrate alone cannot explain this phenomenon.

We utilize antibiotics for treatment in order to establish the relationship between gut microbiota, age, and gout. Different age groups are directly given antibiotics for treatment. We found that after clearing the gut microbiota and then stimulating with MSU, the trend of inflammation factors changing with age disappears.

(2) In Figure 2, the fecal transplantation from young mice improved the infiltration of inflammatory cells and inflammatory cytokines in the Old and Aged groups. However, in Supplementary Figure 1A, there is no improvement observed in the percentage of foot swelling. Is it appropriate to conclude that inflammation was improved even though foot swelling was not suppressed?

Although we did not observe changes in the swelling of the mice's feet, there were changes in the inflammatory cell infiltration and inflammation factors in the slices. We rely on a comprehensive assessment of various indicators to determine whether the inflammatory condition has improved or worsened.

(3) In line #249, the authors state that "the fecal microbiota from mice in the young group promotes uric acid elimination, inhibits reabsorption, and may contribute to the integrity of the intestinal barrier structure." However, Supplementary Figure 3F-H shows no significant alterations in Occludin and ZO-1 mRNA expression levels among all groups. Therefore, it is difficult to conclude that the fecal microbiota from the young group promotes the integrity of the intestinal barrier structure. A functional barrier assay, such as oral administration of FITC-dextran, would be necessary to verify the authors' conclusion.

In Supplementary Figure 3F-H, we observed that the mRNA expression of Occludin and ZO-1 increased but showed no significant difference. However, after the elderly mice were transplanted with the intestinal microbiota of young mice, the mRNA expression of JAMA showed a significant upward trend. Additionally, due to the scarcity of old mice, we were unable to perform the oral administration of FITC-dextran. However, we supplemented with immunohistochemical slices of Zo-1 and Occludin to support our viewpoint.

(4) In Figure 4, when comparing the young+PBS group with the old+PBS or aged+PBS groups, there are hardly any differences in the proteins involved in uric acid synthesis (ADA, GDA, XOD) or the genes involved in uric acid transport (URAT1, GLUT9, OAT1, OTA3, ABCG2). Since no changes in uric acid synthesis or transport pathways are observed with aging, it is questionable to conclude that fecal transplantation from young mice improves these pathways and lowers blood uric acid levels.

In the calculation process, we used different age groups of the control group as references, instead of directly using young mice. We then compared the data of mice of different ages, and the results are in Supplementary Material 4.

(5) In line 276, the authors describe "the Young +Old and Young+Aged groups tended to be closer to the Old+PBS and Aged+PBS groups, and the Old+Young and Aged+young groups tended to be closer to the Young+PBS group (Figure 5D)". Please conduct a statistical analysis.

(6) In line 298, the authors hypothesize that butyrate might be the key molecule responsible for controlling gout, as Bifidobacterium and Akkermansia were abundant in the Young group, and the butyrate pathway was prominent. However, neither Bifidobacterium nor Akkermansia are butyrate-producing bacteria. Thus, the conclusion appears to be biased toward butyrate, raising questions about this interpretation.

Upon comparison, we discovered other bacteria genera that produce butyrate, such as Lachnoclostridium. Additionally, literature (PMID:38126785, 26420851) reports have indicated that Bifidobacteria combined with other genera can enhance the production of butyrate. Meanwhile, Akkermansia, particularly the species Akkermansia muciniphila, has been found to confer several beneficial traits, as evidenced by preclinical studies. These traits include promoting the growth of butyrate-producing bacteria through the production of acetate, which leads to a decrease in the loss of the colonic bilayer and subsequent reduction in inflammation (PMID:35468952). Based on the predicted results of microbiome functions, we observed that the Butanoate_metabolism of the microbiota in young mice and the elderly mice recipients of young mouse microbiota was enhanced. Considering that Lachnoclostridium can produce butyrate, and that Bifidobacteria and Akkermansia can promote the production of butyrate by the intestinal microbiota, we speculated that butyrate might play a role in gout and hyperuricemia.

(7) In Supplementary Figure 7, acetic acid and propionic acid also show the same behavior as butyric acid. It is possible that these metabolites may also affect the development of gout.

Thank you for your suggestion. Indeed, Figure 7 does show a similar trend for acetic and propionic acids as for butyric acid. However, considering the predictive data of microbial function and the non-targeted metabolomic data, there is an enhancement of Butanoate_metabolism in both young mice and elderly mice receiving young mouse intestinal microbiota transplants. Therefore, we prioritized butyrate as the subject of our study. Due to the scarcity of elderly mice, we are unable to conduct subsequent experiments with acetic and propionic acids, which is one of the limitations of this study. This work will be addressed in our follow-up research.

(8) In Figure 6, the secondary bile acid biosynthesis pathway was also changed. However, there is little mention of secondary bile acid in the discussion section. Please carefully discuss other possibilities besides butyrate.

Thank you for your suggestion. We have incorporated a discussion about secondary bile acids into the relevant section of our manuscript.

(9) In line #330, the authors state, 'the metabolites identified as showing differential abundance between the groups were enriched in the butanoate metabolism pathway (Figure 6A-D).' However, there does not appear to be much difference in the butanoate metabolism pathway. Specifically, in Figure 6C, the butanoate metabolism pathway in the Old group does not differ from that in the Young group. Please explain in more detail whether the butanoate metabolism pathway is relevant in the Old group.

The metabolites identified as showing differential abundance between the groups were enriched in the butanoate metabolism pathway. The differential metabolites are enriched in the butyrate metabolism pathway; however, the non-targeted metabolomics did not reveal the extent of their enrichment.

(10) In Figure 7, the authors measured the levels of short-chain fatty acids in the Young and Aged groups. They found butyrate in the feces of mice in the Young group was higher than that in the Aged group. However, I wonder whether the Old group also had low levels of butyrate or not.

In the experiment, we selected three representative groups to verify the hypothesis that butyrate may play a significant role in gout and hyperuricemia. Subsequently, we found that supplementing 18-month-old and 24-month-old mice with butyrate indeed reduced blood uric acid levels and alleviated gout symptoms. Since 18-month-old mice are difficult to obtain, we only conducted microbiome sequencing and non-targeted metabolomic analysis.

Minor issues:

(11) In line 74, what does MSU stand for? Please describe the abbreviation.

In line 74, MSU refers to Monosodium urate crystals.

(12) In line 136, please insert a space between "IL-1β" and "and".

Thank you for your suggestion. We have corrected the error of the article.

(13) In line 570, please describe the method of butyrate administration and also correct the grammatical errors.

Thank you for your suggestion. We have corrected the error of the article.

(14) Change the title of x axis in Figure 2F-H, "Serum ~" to "Peritoneal fluid ~", according to the legend.

Thank you for your suggestion. We have corrected this error in the manuscript.

(15) In line 302, "succinates" should be "butyric acid or butyrate".

Thank you for your suggestion. We have corrected this error in the manuscript.

Reviewer #3 (Recommendations For The Authors):

(1) The authors showed the results of IL-1β levels in foot tissues in Figure 1C and Figure 1H, and serum IL-1β, IL-6, and TNF-α levels in Figure 2F-H. Could the authors also provide the results of IL-6 and TNF-α in foot tissue in Figure 1?

Thank you for your suggestion. We have added the results of of IL-6 and TNF-α in foot tissue in supplementary material 4.

(2) There are some errors in the reference citation format, such as missing page numbers.

Thank you for your careful review. We have revised the references in our manuscript.

(3) There are too many writing errors in the manuscript, which greatly affect the understanding of the text. The manuscript must be carefully revised to improve its readability. It's recommended that a professional English writer or native speaker proofread the paper before submission. Some errors, but not limited to these errors, are listed below.

a. Line 107: The abbreviation for "short-chain fatty acid" should be SCFA, not SFCA.

Thank you for your careful review. We have corrected this error in the manuscript.

b. Line 136: There is a missing space between IL-1β and and. B.

Thank you for your careful review. We have corrected this error in the manuscript.

c. Line 145, the phrase "on gout on gout", and line 471, "that transplantation" are repeated.

Thank you for your careful review. We have corrected this error in the manuscript.

d. Line 152: "Age+PBS" should be "Aged+PBS".

Thank you for your careful review. We have corrected this error in the manuscript.

e. In Figure 1e, "Aded+PBS" should be "Aged+PBS".

Thank you for your careful review. We have corrected the error in Figure 1e.

f. Line 152: The phrase "by via" is repeated.

Thank you for your suggestion. We have deleted the phrase "by via" in line 152.

g. "16S rDNA" in line 92 is inconsistent with the "16S rRNA" in line 652.

Thank you for your suggestion. We have revised the error in the manuscript to maintain consistency in professional terminology.