Neutralizing gut-derived lipopolysaccharide as a novel therapeutic strategy for severe leptospirosis

  1. Xufeng Xie
  2. Xi Chen
  3. Shilei Zhang
  4. Jiuxi Liu
  5. Wenlong Zhang
  6. Yongguo Cao

  • Curated by eLife

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    The gut microbiota influences many infectious diseases; however, its role Leptospirosis remains unclear. In this fundamental work, Xie et al. use a hamster model to show that Leptospira infection leads to gut pathology, an altered gut microbiota, and increased translocation. A combined use of antibiotics and LPS neutralization prolonged survival, providing a potential new therapeutic approach. This study utilizes compelling methods to provide new insights into this emerging disease, which could be dissected further in future studies aimed at gaining mechanistic insight and assessing the translational relevance of these discoveries.

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Abstract

Leptospirosis is an emerging infectious disease caused by pathogenic Leptospira spp. Humans and some mammals can develop severe forms of leptospirosis accompanied by a dysregulated inflammatory response, which often results in death. The gut microbiota has been increasingly recognized as a vital element in systemic health. However, the precise role of the gut microbiota in severe leptospirosis is still unknown. Here, we aimed to explore the function and potential mechanisms of the gut microbiota in a hamster model of severe leptospirosis. Our study showed that leptospires were able to multiply in the intestine, cause pathological injury, and induce intestinal and systemic inflammatory responses. 16S rRNA gene sequencing analysis revealed that Leptospira infection changed the composition of the gut microbiota of hamsters with an expansion of Proteobacteria. In addition, gut barrier permeability was increased after infection, as reflected by a decrease in the expression of tight junctions. Translocated Proteobacteria were found in the intestinal epithelium of moribund hamsters, as determined by fluorescence in situ hybridization, with elevated lipopolysaccharide (LPS) levels in the serum. Moreover, gut microbiota depletion reduced the survival time, increased the leptospiral load, and promoted the expression of proinflammatory cytokines after Leptospira infection. Intriguingly, fecal filtration and serum from moribund hamsters both increased the transcription of TNF-α , IL-1β , IL-10 , and TLR4 in macrophages compared with those from uninfected hamsters. These stimulating activities were inhibited by LPS neutralization using polymyxin B. Based on our findings, we identified an LPS neutralization therapy that significantly improved the survival rates in severe leptospirosis when used in combination with antibiotic therapy or polyclonal antibody therapy. In conclusion, our study not only uncovers the role of the gut microbiota in severe leptospirosis but also provides a therapeutic strategy for severe leptospirosis.

Article activity feed

  1. Version published to 10.7554/elife.96065.3 on eLife
  2. Version published to 10.7554/elife.96065 on eLife
  3. Version published to 10.7554/elife.96065.2 on eLife
  4. eLife

    Author response:

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

    Reviewer #1 (Comments to the Author):

    Summary:

    In this study, Xie and colleagues aimed to explore the function and potential mechanisms of the gut microbiota in a hamster model of severe leptospirosis. The results demonstrated that Leptospira infection was able to cause intestine damage and inflammation. Leptospira infection promoted an expansion of Proteobacteria, increased gut barrier permeability, and elevated LPS levels in the serum. Thus, they proposed an LPS-neutralization therapy which improved the survival rate of moribund hamsters combined with antibody therapy or antibiotic therapy.

    Strengths:

    The work is well-designed and the story is interesting to me. The gut microbiota is essential for immunity and systemic health. Many life-threatening pathogens, such as SARS-CoV-2 and other gut-damaged infection, have the potential to disrupt the gut microbiota in the later stages of infection, causing some harmful gut microbiota-derived substances to enter the bloodstream. It is emphasized that in addition to exogenous pathogenic pathogens, harmful substances of intestinal origin should also be considered in critically ill patients.

    Weaknesses:

    Q1: There are many serotypes of Leptospira, it is suggested to test another pathogenic serotype of Leptospira to validate the proposed therapy.

    That’s a constructive suggestion. We have tested another pathogenic serotype of Leptospira (L. interrogans serovar Autumnalis strain 56606) to verify the LPS-neutralization therapy combined with antibiotic therapy (Supplementary Fig. S9B). The results showed that the combination of the LPS-neutralization therapy with antibody therapy or antibiotic therapy also significantly improved the survival rate of hamsters infected by 56606.

    Q2: Authors should explain why the infective doses of leptospires was not consistent in different study.

    Thank you for your comment. To examine the role of the gut microbiota on acute leptospirosis, the infective doses of leptospires was chosen for 106, while in other sections of the study, the infective doses of leptospires was chosen for 107. In fact, we also used 107 leptospires to infect hamsters, however, the infective doses of 107 leptospires might be overdose, there was no significant difference on the survival rate between the control group and the Abx-treated group. A previous study also highlighted that the infective doses of leptospires was important in the investigating the sex on leptospirosis, as male hamsters infected with L. interrogans are more susceptible to severe leptospirosis after exposure to lower infectious doses than females (103 leptospires but not 104 leptospires) (1).

    Reference

    (1) GOMES C K, GUEDES M, POTULA H H, et al. Sex Matters: Male Hamsters Are More Susceptible to Lethal Infection with Lower Doses of Pathogenic Leptospira than Female Hamsters (J). Infect Immun, 2018, 86(10).

    Q3: In the discussion section, it is better to supplement the discussion of the potential link between the natural route of infection and leptospirosis.

    Thank for your suggestion. We have supplemented it in the discussion (line 523-527 in the track change PDF version).

    Q4: Line 231, what is the solvent of thioglycolate?

    We have supplemented it in the manuscript (line 242-243 in the track change PDF version).

    Q5: Lines 962-964, there are some mistakes which are not matched to Figure 7.

    Thank you for pointing that out, we have corrected it in the manuscript.

    Reviewer #2 (Comments to the Author):

    Summary:

    Severe leptospirosis in humans and some mammals often meet death in the endpoint. In this article, authors explored the role of the gut microbiota in severe leptospirosis. They found that Leptospira infection promoted a dysbiotic gut microbiota with an expansion of Proteobacteria and LPS neutralization therapy synergized with antileptospiral therapy significantly improved the survival rates in severe leptospirosis. This study is well-organized and has potentially important clinical implications not only for severe leptospirosis but also for other gut-damaged infections.

    Weaknesses:

    Q1: In the Introduction section and Discussion section, the authors should describe and discuss more about the differences in the effect of Leptospira infection between mice and hamsters, so that the readers can follow this study better.

    Thank you for your suggestion, we have supplemented it in the manuscript (line 62-66 in the track change PDF version).

    Q2: Lines 92-95, the authors should explain why they chose two different routines of infection.

    Thank you for your comment, we have explained it in the manuscript (line 100 in the track change PDF version).

    Q3: Line 179-180, the concentration of PMB and Dox is missed, and 0.016 μg/L is just ok.

    We have corrected it in the manuscript.

    Q4: "μL" or "μl" and "mL" or "ml' should be uniform in the manuscript.

    Thank you for your suggestion, we have revised it in the manuscript.

    Q5: In the culture of primary macrophages, how many cells are inoculated in the plates should be described clearly.

    We have supplemented it in the manuscript (line 250 in the track change PDF version).

    Q6: Line 271, it is better to list primers used for leptospiral detection in the text. Because it allows readers to find the information they need more directly.

    Thank you for your suggestions, we have supplemented it in the manuscript (line 281-284 in the track change PDF version).

    Q7: Line 366-369, Lactobacillus seems to be a kind of key bacteria during Leptospira infection. A previous study (doi: 10.1371/journal.pntd.0005870) also demonstrated that pre-treatment with Lactobacillus plantarum prevented severe pathogenesis in mice. The authors should discuss the potential probiotic for leptospirosis prevention.

    We have discussed it in the manuscript (line 564-566 in the track change PDF version).

    Q8: Lines 450-451, not all concentrations of fecal filtration from two groups upregulated all gene expression mentioned in the text, the authors should correct it.

    Thank you for pointing that out, we have corrected it in the manuscript (line 461-462 in the track change PDF version).

    Reviewer #3 (Comments to the Author):

    Summary:

    This is a well-prepared manuscript that presented interesting research results. The only defect is that the authors should further revise the English language.

    Strengths:

    The omics method produced unbiased results.

    Weaknesses:

    Q1: LPS neutralization is not a new method for treating leptospiral infection.

    Thank you for your comment. Yes, LPS neutralization is not a new method for treating leptospiral infection, most of which might focus on leptospiral LPS. In addition, Leptospira seemed to be naturally resistant to polymyxin B (1). Recently, neutralizing gut-derived LPS was applied in other diseases which significantly relieved diseases (2-3). In this study, we found that Leptospira infection promoted an expansion of Proteobacteria, increased gut barrier permeability, and elevated LPS levels in the serum. Thus, we proposed an LPS-neutralization therapy which improved the survival rate of moribund hamsters combined with antibody therapy or antibiotic therapy.

    Reference

    (1) LIEGEON G, DELORY T, PICARDEAU M. Antibiotic susceptibilities of livestock isolates of leptospira (J). Int J Antimicrob Agents, 2018, 51(5):693-699.

    (2) MUNOZ L, BORRERO M J, UBEDA M, et al. Intestinal Immune Dysregulation Driven by Dysbiosis Promotes Barrier Disruption and Bacterial Translocation in Rats With Cirrhosis (J). Hepatology, 2019, 70(3):925-938.

    (3) ZHANG X, LIU H, HASHIMOTO K, et al. The gut-liver axis in sepsis: interaction mechanisms and therapeutic potential (J). Crit Care, 2022, 26(1):213.

    Q2: The authors should further revise the English language used in the text.

    Thank you for your suggestion, our manuscript has been polished by American Journal Experts (certificate number: 81C8-C5C1-9D5D-109D-3F23).

  5. eLife

    eLife assessment

    The gut microbiota influences many infectious diseases; however, its role Leptospirosis remains unclear. In this fundamental work, Xie et al. use a hamster model to show that Leptospira infection leads to gut pathology, an altered gut microbiota, and increased translocation. A combined use of antibiotics and LPS neutralization prolonged survival, providing a potential new therapeutic approach. This study utilizes compelling methods to provide new insights into this emerging disease, which could be dissected further in future studies aimed at gaining mechanistic insight and assessing the translational relevance of these discoveries.

  6. eLife

    Reviewer #1 (Public Review)

    Summary:

    In this study, Xie and colleagues aimed to explore the function and potential mechanisms of the gut microbiota in a hamster model of severe leptospirosis. The results demonstrated that Leptospira infection was able to cause intestine damage and inflammation. Leptospira infection promoted an expansion of Proteobacteria, increased gut barrier permeability, and elevated LPS levels in the serum. Thus, they proposed an LPS-neutralization therapy which improved the survival rate of moribund hamsters combined with antibody therapy or antibiotic therapy.

    Strengths:

    The work is well-designed and the story are interesting to me. The gut microbiota is essential for immunity and systemic health. Many life-threatening pathogens, such as SARS-CoV-2 and other gut-damaged infection, have the potential to disrupt the gut microbiota in the later stages of infection, causing some harmful gut microbiota-derived substances to enter the bloodstream. It is emphasized that in addition to exogenous pathogenic pathogens, harmful substances of intestinal origin should also be considered in critically ill patients.

  7. eLife

    Reviewer #2 (Public Review):

    Severe leptospirosis in humans and some mammals often meet death in the endpoint. In this article, authors explored the role of the gut microbiota in severe leptospirosis. They found that Leptospira infection promoted a dysbiotic gut microbiota with an expansion of Proteobacteria and LPS neutralization therapy synergized with antileptospiral therapy significantly improved the survival rates in severe leptospirosis. This study is well-organized and has potentially important clinical implications not only for severe leptospirosis but also for other gut-damaged infections.

  8. eLife

    Reviewer #3 (Public Review):

    Summary:

    This is a well prepared manuscript which presented interesting research result.

    Strengths:

    The omics method produced unbiased results.

    Weaknesses:

    LPS neutralization is not new method for treating leptospiral infection.

  9. Version published to 10.1101/2024.01.17.576119v2 on bioRxiv
  10. Version published to 10.7554/elife.96065.1 on eLife
  11. eLife

    eLife assessment

    Herein, Xie and colleagues use a hamster model to show that Leptospira infection leads to gut pathology, an altered gut microbiota, and increased translocation. A combined use of antibiotics and LPS neutralization prolonged survival, providing a potential new therapeutic approach. This fundamental study uses compelling methods to provide new insights into this emerging disease, which could be dissected further in future studies aimed at gaining mechanistic insight and assessing the translational relevance of these discoveries.

  12. eLife

    Reviewer #1 (Public Review):

    Summary:

    In this study, Xie and colleagues aimed to explore the function and potential mechanisms of the gut microbiota in a hamster model of severe leptospirosis. The results demonstrated that Leptospira infection was able to cause intestine damage and inflammation. Leptospira infection promoted an expansion of Proteobacteria, increased gut barrier permeability, and elevated LPS levels in the serum. Thus, they proposed an LPS-neutralization therapy which improved the survival rate of moribund hamsters combined with antibody therapy or antibiotic therapy.

    Strengths:

    The work is well-designed and the story is interesting to me. The gut microbiota is essential for immunity and systemic health. Many life-threatening pathogens, such as SARS-CoV-2 and other gut-damaged infection, have the potential to disrupt the gut microbiota in the later stages of infection, causing some harmful gut microbiota-derived substances to enter the bloodstream. It is emphasized that in addition to exogenous pathogenic pathogens, harmful substances of intestinal origin should also be considered in critically ill patients.

    Weaknesses:

    (1) There are many serotypes of Leptospira, it is suggested to test another pathogenic serotype of Leptospira to validate the proposed therapy.

    (2) Authors should explain why the infective doses of leptospires was not consistent in different study.

    (3) In the discussion section, it is better to supplement the discussion of the potential link between the natural route of infection and leptospirosis.

    (4) Line 231, what is the solvent of thioglycolate?

    (5) Lines 962-964, there are some mistakes which are not matched to Figure 7.

  13. eLife

    Reviewer #2 (Public Review):

    Severe leptospirosis in humans and some mammals often meet death in the endpoint. In this article, authors explored the role of the gut microbiota in severe leptospirosis. They found that Leptospira infection promoted a dysbiotic gut microbiota with an expansion of Proteobacteria and LPS neutralization therapy synergized with antileptospiral therapy significantly improved the survival rates in severe leptospirosis. This study is well-organized and has potentially important clinical implications not only for severe leptospirosis but also for other gut-damaged infections.

  14. eLife

    Reviewer #3 (Public Review):

    Summary:

    This is a well-prepared manuscript that presented interesting research results. The only defect is that the authors should further revise the English language.

    Strengths:

    The omics method produced unbiased results.

    Weaknesses:

    LPS neutralization is not a new method for treating leptospiral infection.

  15. Version published to 10.1101/2024.01.17.576119v1 on bioRxiv