Prophylactic Lipoxin A 4 Attenuates Clostridioides difficile Infection by Augmenting Epithelial Barrier and Resolving Inflammation

  1. Hui Wen
  2. Yunqing Xiang
  3. Yue Yu
  4. Zhixin Ma
  5. Ying Xin
  6. Yufang Deng
  7. Huipai Peng
  8. Yong Shi
  9. Nan Li
  10. Shuqiang Huang

  • Curated by eLife

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    eLife Assessment

    The authors developed and validated a gut-on-chip system to mimic the gut environment for studies of Clostridioides difficile infection in vitro. Although the data generated is useful to the field, the evidence provided to support the conclusions is incomplete. Methodology that is not complete, as well as discrepancies regarding the proposed mode of action of lipoxin A4, are significant weaknesses.

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Abstract

Clostridioides difficile infection (CDI) is a leading healthcare-associated diarrhea with high recurrence rates, partially due to antibiotic-induced dysbiosis and dysregulated host inflammation. Specialized pro-resolving mediators (SPMs), such as Lipoxin A 4 (LXA 4 ), offer promise in controlling excessive inflammation and promoting tissue repair, yet their role in CDI remains unexplored. Here, we developed a compact, gas-tight gut-on-a-chip (GOC) system that reconciles the anaerobic requirements of C. difficile with the oxic needs of human intestinal epithelium, enabling physiologically relevant co-culture within a standard incubator. A CDI in vitro model was established based on this GOC system. Using the model, we demonstrated that prophylactic administration of LXA 4 significantly preserved epithelial barrier integrity, attenuated pro-inflammatory cytokine secretion (IL-8 and IFN-γ), and reduced bacterial colonization. Transcriptomic analysis revealed that LXA 4 pretreatment upregulated genes involved in cell junction organization while downregulated immune activation pathways. These protective effects were validated in a murine CDI model, where LXA 4 pretreatment reduced weight loss, pathological damage, and fecal bacterial burden. Furthermore, prophylactic administration of LXA 4 synergized with vancomycin treatment further enhanced antibiotic efficacy while allowing a 50% dose reduction without compromising therapeutic outcomes. Our study establishes a robust approach for CDI research and highlights the prophylactic and adjuvant potential of inflammation-resolving strategies, offering a novel approach to mitigate CDI incidence and improve treatment outcomes.

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  1. eLife

    eLife Assessment

    The authors developed and validated a gut-on-chip system to mimic the gut environment for studies of Clostridioides difficile infection in vitro. Although the data generated is useful to the field, the evidence provided to support the conclusions is incomplete. Methodology that is not complete, as well as discrepancies regarding the proposed mode of action of lipoxin A4, are significant weaknesses.

  2. eLife

    Reviewer #1 (Public review):

    Summary:

    This study investigates the potential for the immune mediator, lipoxin A4 (LXA4), to alleviate inflammation/damage caused by the healthcare-associated pathogen, Clostridioides difficile. Using both a novel in vitro "gut-on-a-chip" system and a murine model of disease, the authors demonstrate potential disease attenuation by LXA4. Specifically, LXA4 at select administration times during development of C. difficile infection (CDI) may upregulate markers associated with intestinal barrier integrity (ZO-1) and attenuate immune markers typically associated with inflammation (IL-8, IFN-γ, etc.). Overall strengths of the study include the establishment of a novel in vitro model that incorporates anaerobic and aerobic environmental conditions of the gut, as well as some results suggesting a potential role for LXA4 in modulating CDI. However, critical weaknesses of the manuscript, including incomplete methods and a lack of some critical controls or measurements, lead to only partial support for the authors' conclusions. Collectively, the data suggest alternate potential (and perhaps more likely) mechanisms by which LXA4 might modulate CDI. Specific strengths and weaknesses are listed below.

    Strengths:

    (1) A major strength of the study is the use and description of the gastight, gut-on-a-chip system that allows for co-culture of host cells (with aerobic needs) with anaerobic bacteria. While perhaps this (and other in vitro) system does not exactly "more accurately recapitulate specific host-microbe interactions (line 82)", integration of oxic and anoxic conditions that recapitulate the gut is indeed difficult to incorporate in vitro. Results surrounding C. difficile and Caco-2 cell viability in the described system seem substantiated.

    (2) Assessing LXA4 in both an in vitro and in vivo (mouse) model is a complementary strategy. Results from both experiments seem to support the observation that LXA4 can possibly attenuate C. difficile.

    (3) Overall, the manuscript is well-written and straightforward (albeit lacking in some details-see below).

    Weaknesses:

    (1) A major weakness of the manuscript in its current state is that the methods are incomplete or unclear. Details on how C. difficile was handled (strain info, preparation in experiments, quantification) are lacking. Mouse model information (inoculation, housing, number of animals) is missing, particularly for the second set of mouse experiments, which is not described at all in the methods. An IACUC or similar statement is not included.

    a) For in vitro experiments, how exactly were C. difficile quantified using flow cytometry? This is not exactly clear in the methods or the results, where C. difficile counts are referred to as 'normalized' without specific units (Figure 1D). What are these counts normalized to? How much of the total effluent was measured? This might also explain the discrepancy in C. difficile counts, referred to below.

    b) How exactly were C. difficile quantified for the mouse studies? The authors state that fecal samples were plated on CCFA agar, but the y-axis merely states "numbers of bacteria". Other bacteria grow on CCFA. How were C difficile specifically enumerated?

    c) Figure 4. For the vancomycin / LXA4 experiments, were mice subjected to antibiotics to render them initially susceptible to C. difficile? If so, this should be included in experimental timelines. If not, how do the investigators know that mice were colonized with C. difficile in each instance (usually mice require abx perturbation for susceptibility)? How was vancomycin administered to mice? In any case, C. difficile loads should be quantified for all conditions in these experiments.

    d) Related to the above (Figure 4 experiments), were all of these measurements taken only 24 hours post-infection? These experiments are not described well in the results and are not described at all in the methods.

    e) How many total mice were included in the study groups, and how were they housed? Cage effects can influence any mouse study, but are especially important in CDI studies, given the importance of the microbiome in the development of CDI.

    f) How were mice inoculated with C. difficile? Was this a spore or vegetative inoculum, and how? The state inoculum of 1x10^-9 is quite large.

    g) What is the history/ribotype of the C. difficile strain (1482?) used in all the experiments? How does this compare to other commonly used strains of C. difficile? Different strains demonstrate overall virulence, disease dynamics, and disease severity in animal and in vitro models.

    (2) Related to some methodological clarifications, there are some missing controls that would bolster support for final interpretations and some odd discrepancies in the study that are not explained.

    a) Figure 1C: How does the mucin layer (i.e., Caco-2 cell differentiation) look under anoxic conditions? This measurement was only included in the oxic conditions.

    b) In initial C. difficile quantification within the system (Figure 1D), C. difficile counts seem to range from 3 - 12 (undefined units). In the C. difficile / LXA4 experiments, these counts only reach ~1.8 (undefined units) in the CDI group. What explains this large discrepancy? Furthermore, the prophylactic LXA4 group seems to hover around < 0.5, similar to what is seen at 0 or 3 hours with C. difficile alone. This suggests that C. difficile might not proliferate at all in the presence of LXA4, perhaps explaining why epithelial barrier functions and immune attenuation are improved.

    c) Figure 2B. What do untreated controls (no CDI, but with or without LX4A) look like compared to the experimental groups? These controls should be included with the main Figure 2 results.

    d) If all metrics in Figure 4 were measured only 24 hours after infection, this is a VERY short timeline for CDI. Depending on the strain, damage might not even be quantifiable by this time point. For instance, C. difficile 630 disease signs only appear 2-4 days post-infection. C. difficile VPI kills mice within 36 hours, but Figure 3 results suggest that mice survive just fine. What is known about this strain's disease dynamics in mice? Alternatively, is it possible that LXA4 alone increases barrier integrity / attenuates inflammation? The inclusion of non-CDI controls (with or without abx; untreated; etc) might help distinguish this.

    (3) Perhaps the largest weakness of the manuscript is the interpretation of how LXA4 might attenuate CDI, which is also misleading as a title. The authors purport that disease attenuation is via LXA4, increasing barrier integrity and attenuating inflammation. However, much of the evidence suggests that LXA4 might limit C. difficile colonization. If there is less C. difficile (thus less toxin) in any system, all aspects of the disease will be attenuated. Indeed, their data suggest that there are decreasing amounts of C. difficile in the presence of LXA4, which could be due to direct inhibition of C. difficile or its toxin, removing nutrients necessary for C. difficile growth, or indirect effects on microbes in the gut environment (in mice). Proper quantification of C. difficile, toxin measurements, and dose responses would better distinguish which mechanism is more likely.

    a) The initial LXA4 experiments assessing potential therapeutic effects (mainly Figure 2) were conducted at 6 hours post-infection. What is the C. difficile load and/or toxin burden at this time? In some ways, LXA4 administration at this time point could also be thought of as 'prophylactic', given that damage (and maybe C. difficile virulence?) has not occurred yet.

    b) Is it possible that LX4A administration prior to C. difficile inoculation influences C. difficile physiology (colonization; toxin production), rather than alleviating C. difficile damage? C. difficile colonization should be quantified in all the LX4A experiments (only a subset is shown in Figure 2).

    c) Line 213 / Figure 2G. While it is possible that "LXA4 reprograms the intestinal epithelial transcriptome to bolster barrier function and temper immune signaling", the decreased C. difficile measurements in the presence of LXA4 suggest it impacts C. difficile colonization / function. This decreased level of C. difficile (and thus less toxin) could also explain immune response attenuation. Toxin measurements, as well as some C. difficile dose responses within the system, could help distinguish which possibility is more likely.

    d) Both in vitro and in vivo experimental results suggest a prophylactic role for LXA4 in CDI. However, the current experiments cannot distinguish whether this prophylactic response is due to host-specific anti-inflammatory attenuation (which the authors suggest) or due to an impact on C. difficile colonization/function (which is not acknowledged). The effect of LXA4 on C. difficile could be via direct inhibition of C. difficile growth or host remodeling that modulates C. difficile colonization or metabolism.

    e) Figure 4. While the data seem to support some preservation of barrier function and attenuation of inflammatory responses, this could once again be due to delaying, decreasing, or inhibiting C. difficile colonization itself, rather than attenuation by LXA4. Indeed, vancomycin-induced improvements within this short amount of time are likely due to inhibiting C. difficile, as it is an antibiotic used to directly kill C. difficile.

    (4) Other comments:

    a) Given that the current results cannot preclude alternate, if not more likely, explanations for how LXA4 might attenuate CDI, the manuscript should include a more comprehensive discussion. This could include study caveats, C. difficile-specific context about infection (i.e., infection dynamics, context with other experiments).

    b) Dysbiosis: undefined definition, as this is context-dependent. For CDI, what does this mean?

    c) Unclear if in vitro intestinal models "more accurately recapitulate specific host-microbe interactions", even considering caveats of animal models. Rather, each model has their own purpose; I would be careful about this phrasing (line 82).

    d) Line 86: not just "thrives under strict anaerobic conditions", but is necessary for growth. C. difficile is an obligate anaerobe.

  3. eLife

    Reviewer #2 (Public review):

    C. difficile infection (CDI) is one of the most common nosocomial intestinal infections with a high rate of disease recurrence. Importantly, antibiotics used to treat CDI are a double-edged sword because disruption of the gut microbiome also increases the susceptibility to CDI. Therefore, there is an unmet need for alternative therapeutic approaches against CDI. CDI pathogenesis is initiated by the cytotoxic toxins TcdA and TcdB that target and induce cell death of intestinal epithelial cells, leading to epithelial barrier breakdown and inflammation. Innate immune cells such as neutrophils and innate lymphoid cells (ILCs) were shown to be crucial to control CDI during the acute phase. Based on previous reports that the pro-resolving mediator Lipoxin A4 (LXA4) inhibits neutrophil infiltration and promotes efferocytosis as well as mucosal repair, the authors reason that LXA4 could be leveraged as a therapy against CDI.

    The authors developed and validated a gut-on-chip (GOC) system to mimic the gut environment for C. difficile infection in vitro studies. LXA4 was able to decrease C. difficile-induced inflammation only when used as a prevention but not as a therapy. IEC RNA-seq revealed that LXA4 treatment upregulates a transcriptional program that reinforces barrier function. These data were replicated in an in vivo model of CDI. Overall, the study provides evidence that LXA4 could be repurposed for CDI treatment, but some claims are not fully supported by the data, such as the synergy between LXA4 and vancomycin, which has not been experimentally tested in vivo.

  4. eLife

    Author response:

    Public Reviews:

    Reviewer #1 (Public review):

    (1) Completeness and clarity of Methods (Weakness #1).

    We will substantially expand the Methods section to include:

    (a) Detailed information on C. difficile strain ribotype 1382 (correcting the typographical error "1482"), including its virulence characteristics, toxin production dynamics, and rationale for its selection.

    (b) Step-by-step protocols for on-chip bacterial quantification by flow cytometry, including sample collection volume, processing, and the specific normalization procedure (with clarification that normalized values are intended for within-experiment comparisons only).

    (c) Full description of mouse experiments: antibiotic pre-treatment regimen, inoculation details (spores vs. vegetative cells, justification of the 1×10^9 CFU dose), animal numbers, housing conditions, and cage-effect considerations. The IACUC approval statement will be moved from Acknowledgments to Methods.

    (2) Mucin layer characterization under anoxia (Weakness #2a).

    We will clarify in the Methods that mucin staining was performed after the initial oxic culture phase to confirm differentiation prior to anaerobic challenge. We will cite relevant literature discussing the stability of pre-formed mucin layers under short-term anoxic conditions and incorporate this discussion to contextualize our experimental design in the revised Methods.

    (3) Discrepancy in C. difficile counts and mechanism of LXA4 action (Weakness #2b, #3).

    We will provide a detailed explanation of our flow cytometry normalization algorithm, emphasizing that values are only comparable within a given experimental batch. We plan to perform additional in vitro experiments to directly assess the effect of LXA4 on bacterial growth and toxin secretion. These data will help distinguish between direct antibacterial effects and host-mediated protection, and the revised Discussion will incorporate this analysis.

    (4) Missing controls and experimental timelines (Weakness #2c–d).

    We will clarify that Figure 4 presents gut-on-chip experiments, not animal studies. The corresponding methods will be fully described. Additionally, we will include cross-experiment alignment analyses (using the CDI group as a common reference) to integrate negative control data from separate experimental batches. We also plan to generate additional data examining the effect of LXA4 alone (without infection) on epithelial barrier integrity and inflammatory status, which will be included as supplementary controls.

    (5) C. difficile strain characterization (Weakness #1g).

    A comprehensive section on ribotype 1382 will be added to the Methods, detailing its in vitro growth kinetics, toxin production profiles, and disease dynamics in the murine model, with appropriate literature citations.

    (6) Dysbiosis definition and phrasing adjustments (Other comments #b–d).

    We will revise the text to provide a clear definition of dysbiosis in the context of CDI. We will also temper the phrasing in line 82 to more accurately describe the advantages of our GOC system relative to other in vitro models, and correct the description of C. difficile as an obligate anaerobe.

    Reviewer #2 (Public review):

    (1) Synergy between LXA4 and vancomycin in vivo.

    We agree that the synergistic effect observed in the GOC model requires validation in an animal model. We are currently conducting mouse experiments to test the combination of prophylactic LXA4 with vancomycin treatment. The results will be included as a new Figure 5 in the revised manuscript.

    We are confident that these planned revisions will fully address the reviewers' concerns and significantly enhance the rigor and impact of our study.

  5. Version published to 10.64898/2026.02.03.703554 on bioRxiv