Secretory leukocyte protease inhibitor influences periarticular joint inflammation in B. burgdorferi-infected mice
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eLife Assessment
This study presents a valuable finding on the role of secretory leukocyte protease inhibitors (SLPI) in developing Lyme disease in mice infected with Borrelia burgdorferi. The evidence supporting the authors' claims is solid. However, several concerns raised by the reviewers remain unaddressed. This paper would be of interest to scientists in the infectious inflammatory disease field.
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Abstract
Lyme disease, caused by Borrelia burgdorferi , is the most common tick-borne infection in the United States. Arthritis is a major clinical manifestation of infection, and synovial tissue damage has been attributed to the excessive pro-inflammatory responses. The secretory leukocyte protease inhibitor (SLPI) promotes tissue repair and exerts anti-inflammatory effects. The role of SLPI in the development of Lyme arthritis in C57BL/6 mice, which can be infected with B. burgdorferi , but only develop mild joint inflammation, was therefore examined. SLPI -deficient C57BL/6 mice challenged with B. burgdorferi had a higher infection load in the tibiotarsal joints and marked periarticular swelling, compared to infected wild type control mice. The ankle joint tissues of B. burgdorferi- infected SLPI -deficient mice contained significantly higher percentages of infiltrating neutrophils and macrophages. B. burgdorferi -infected SLPI -deficient mice also exhibited elevated serum levels of IL-6, neutrophil elastase, and MMP-8. Moreover, using a recently developed BASEHIT ( BA cterial S election to E lucidate H ost-microbe I nteractions in high T hroughput) library, we found that SLPI directly interacts with B. burgdorferi . These data demonstrate the importance of SLPI in suppressing periarticular joint inflammation in Lyme disease.
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eLife Assessment
This study presents a valuable finding on the role of secretory leukocyte protease inhibitors (SLPI) in developing Lyme disease in mice infected with Borrelia burgdorferi. The evidence supporting the authors' claims is solid. However, several concerns raised by the reviewers remain unaddressed. This paper would be of interest to scientists in the infectious inflammatory disease field.
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Reviewer #2 (Public review):
This study by Yu and coworkers investigates the potential role of Secretory leukocyte protease inhibitor (SLPI) in Lyme arthritis. They show that, after needle inoculation of the Lyme disease agent, B. burgdorferi, compared to wild type mice, a SLPI-deficient mouse suffers elevated bacterial burden, joint swelling and inflammation, pro-inflammatory cytokines in the joint, and levels of serum neutrophil elastase (NE). They suggest that SLPI levels of Lyme disease patients are diminished relative to healthy controls. Finally, using a powerful screen of secreted mammalian proteins, they find that SLPI interacts directly B. burgdorferi.
The known role of SLPI in dampening inflammation and inflammatory damage by inhibition of NE makes the enhanced inflammation in the joint of B. burgdorferi-infected mice a …
Reviewer #2 (Public review):
This study by Yu and coworkers investigates the potential role of Secretory leukocyte protease inhibitor (SLPI) in Lyme arthritis. They show that, after needle inoculation of the Lyme disease agent, B. burgdorferi, compared to wild type mice, a SLPI-deficient mouse suffers elevated bacterial burden, joint swelling and inflammation, pro-inflammatory cytokines in the joint, and levels of serum neutrophil elastase (NE). They suggest that SLPI levels of Lyme disease patients are diminished relative to healthy controls. Finally, using a powerful screen of secreted mammalian proteins, they find that SLPI interacts directly B. burgdorferi.
The known role of SLPI in dampening inflammation and inflammatory damage by inhibition of NE makes the enhanced inflammation in the joint of B. burgdorferi-infected mice a predicted result but it has not previously been demonstrated and could spur further study. A limitation that is unaddressed experimentally is potential contribution of the greater bacterial burden to the enhanced inflammation, leaving open the question of whether greater immunologic stimulus or a defect in the regulation of inflammation is responsible for the observed enhanced disease. Answering this question would better justify the statement in the abstract that "These data demonstrate the importance of SLPI in suppressing periarticular joint inflammation in Lyme disease."
Although the finding of SLPI binding to bacteria is potentially quite interesting the biological relevance of this interaction is not addressed. Readers of only the abstract, which describes the direct interaction of SLPI with bacteria, may mistakenly conclude that the authors demonstrate that recruitment of this immunoregulatory factor to the bacterial surface enhances inflammation of infected tissues. This attractive possibility has not been demonstrated in this study; such assertion would require comparison of bacteria that either bind or do not bind SLPI in a mouse infection model.
Finally, the investigators take advantage of clinical samples to ask if serum SLPI levels a diminished in Lyme disease patients relative to healthy controls. The assessment of human samples is interesting and generally to be lauded, but here the comparison is limited by: (a) a small sample number, with only 5 healthy control samples (which should not be difficult to obtain); and (b) the inclusion of samples from 4 patients with erythema migrans rather than Lyme arthritis, which was the manifestation tracked in the mouse studies. Moreover, of the 3 Lyme arthritis patients, serum samples from multiple blood draws were included, resulting in 5 data points; similarly, of the 4 erythema migrans patients, 13 separate samples were included. The multiple samplings from some but not all subjects could result in differential "weighting" of samples. Therefore, although the investigators provide a statistical analysis of these data, it is difficult to evaluate the validity of this apparent difference.
In summary, this is an interesting study that provides new information regarding infection in a host deficient in SLPI and, using a state-of-the-art screen of the mammalian secretome to show that B. burgdorferi binds SLPI, raising the attractive possibility that this pathogen utilizes a host immune regulator to enhance inflammation. The conclusions that SLPI enhances inflammation directly due to its immunoregulatory activity and that SLPI levels are diminished in human Lyme disease patients, as well as the implication that SLPI binding by the bacterium has pathogenic significance, each require further study.
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Author response:
The following is the authors’ response to the current reviews.
We deeply appreciate the reviewer’s careful review and critiques. These are excellent critiques that we are working on and probably require a few more years of work. Published together, we believe these critiques add value to our manuscript.
The following is the authors’ response to the original reviews.
Reviewer #2 (Public review):
Summary:
This manuscript by Yu and coworkers investigates the potential role of Secretory leukocyte protease inhibitor (SLPI) in Lyme arthritis. They show that, after needle inoculation of the Lyme disease (LD) agent, B. burgdorferi, compared to wild type mice, a SLPI-deficient mouse suffers elevated bacterial burden, joint swelling and inflammation, pro-inflammatory cytokines in the joint, and levels of serum neutrophil …
Author response:
The following is the authors’ response to the current reviews.
We deeply appreciate the reviewer’s careful review and critiques. These are excellent critiques that we are working on and probably require a few more years of work. Published together, we believe these critiques add value to our manuscript.
The following is the authors’ response to the original reviews.
Reviewer #2 (Public review):
Summary:
This manuscript by Yu and coworkers investigates the potential role of Secretory leukocyte protease inhibitor (SLPI) in Lyme arthritis. They show that, after needle inoculation of the Lyme disease (LD) agent, B. burgdorferi, compared to wild type mice, a SLPI-deficient mouse suffers elevated bacterial burden, joint swelling and inflammation, pro-inflammatory cytokines in the joint, and levels of serum neutrophil elastase (NE). They suggest that SLPI levels of Lyme disease patients are diminished relative to healthy controls. Finally, they find that SLPI may interact directly the B. burgdorferi.
Strengths:
Many of these observations are interesting and the use of SLPI-deficient mice is useful (and has not previously been done).
Weaknesses:
(a) The known role of SLPI in dampening inflammation and inflammatory damage by inhibition of NE makes the enhanced inflammation in the joint of B. burgdorferi-infected mice a predicted result; (b) The potential contribution of the greater bacterial burden to the enhanced inflammation is acknowledged but not experimentally addressed; (c) The relationship of SLPI binding by B. burgdorferi to the enhanced disease of SLPI-deficient mice is not addressed in this study, making the inclusion of this observation in this manuscript incomplete; and (d) assessment of SLPI levels in healthy controls vs. Lyme disease patients is inadequate.
We greatly appreciate the critiques, and we do agree. Even though the observation of NE level is predictable, we believe that it is important to actually demonstrate it in the context of murine Lyme arthritis. The function of SLPI goes beyond inhibiting NE level. As an ongoing project in our lab, we believe that the current study serves as a good starting point to explore the pleiotropic effects SLPI in the pathogenesis of murine Lyme arthritis and in patients. And, the critiques here are of great value to our research.
Comments on revised version:
Several of the points were addressed in the revised manuscript, but the following issues remain:
Previous point that the relationship of SLPI binding to B. burgdorferi to the enhanced disease of SLPI-deficient mice is not investigated: The authors indicate that such investigations are ongoing. In the absence of any findings, I recommend that their interesting BASEHIT and subsequent studies be presented in a future study, which would have high impact.
We thank the reviewer for the critique. We do agree that this part of the story is not complete. However, we would like to keep the BASEHIT and binding data in the paper, as we believe that it is an important finding. We confirmed the binding using ELISA, flow cytometry, and immunofluorescent microscopy. We showed that the binding is specific to infectious strain of B. burgdorferi, thus likely to contribute to the pathogenesis of murine Lyme arthritis. Our data suggest that SLPI can directly interact with a B. burgdorferi protein. We are exploring the biological significance of the binding. And this finding can be further explored by other labs too.
Previous recommendation 1: (The authors added lines 267-68, not 287-68). This ambiguity is acknowledged but remains. In addition, in the revised manuscript, the authors state "However, these data also emphasize the importance of SLPI in controlling the development of inflammation in periarticular tissues of B. burgdorferi-infected mice." Given acknowledged limitations of interpretation, "suggest" would be more appropriate than "emphasize".
We thank the reviewer for the careful reading, and we apologize for the mistake. The change has been made accordingly (line 268).
Previous recommendation 5: The lack of clinical samples can be a challenge. Nevertheless, 4 of the 7 samples from LD patients are from individuals suffering from EM rather than arthritis (i.e., the manifestation that is the topic of the study) and some who are sampled multiple times, make an objective statistical comparison difficult. I don't have a suggestion as to how to address the difference in number of samples from a given subject. However, the authors could consider segregating EM vs. LA in their analysis (although it appears that limiting the comparison between HC and LA patients would not reveal a statistical difference).
We thank the reviewer for the critique. And we agree with the reviewer that the patient’s data presented are not ideal. We believe that at this point the combination of the samples is most logical, as the number of samples we have from patients with Lyme arthritis is fairly limited. We stated the limitation in the discussion. We do believe that the finding of the correlation is important. It suggests the potential function of SLPI in patients, beyond murine infection.
What’s more, various groups with large number of different samples can elucidate the relationship further.
Previous recommendation 6: Given that binding of SLPI to the bacterial surface is an essential aspect of the authors' model, and that the ELISA assay to indicate SLPI binding used cell lysates rather than intact bacteria, a control PI staining to validate the integrity of bacteria seems reasonable.
We appreciate the suggestion and has provided the propidium iodide staining in Supplemental Figure 5 (line 539-542, 568-569, 718-722).
Previous recommendation 8: The inclusion of a no serum control (that presumably shows 100% viability) would validate the authors' assertion that 20% serum has bactericidal activity.
We appreciate the suggestion. As stated in the manuscript (line 583-584), the percent viability was normalized to the control spirochetes culture without any treatment. Thus, the control spirochetes culture, without serum and SLPI treatment, showed 100% viability. We have revised Supplemental Figure 3 accordingly.
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eLife Assessment
This study presents a valuable finding on the role of secretory leukocyte protease inhibitors (SLPI) in developing Lyme disease in mice infected with Borrelia burgdorferi. The evidence supporting the claims of the authors is solid. This paper will be of interest to scientists in the infectious inflammatory disease field.
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Reviewer #1 (Public review):
Summary:
This study demonstrates the significant role of secretory leukocyte protease inhibitor (SLPI) in regulating B. burgdorferi-induced periarticular inflammation in mice. They found that SLPI-deficient mice showed significantly higher B. burgdorferi infection burden in ankle joints compared to wild-type controls. This increased infection was accompanied by infiltration of neutrophils and macrophages in periarticular tissues, suggesting SLPI's role in immune regulation. The authors strengthened their findings by demonstrating a direct interaction between SLPI and B. burgdorferi through BASEHIT library screening and FACS analysis. Further investigation of SLPI as a target could lead to valuable clinical applications.
The conclusions of this paper are mostly well supported by data. And the authors were …
Reviewer #1 (Public review):
Summary:
This study demonstrates the significant role of secretory leukocyte protease inhibitor (SLPI) in regulating B. burgdorferi-induced periarticular inflammation in mice. They found that SLPI-deficient mice showed significantly higher B. burgdorferi infection burden in ankle joints compared to wild-type controls. This increased infection was accompanied by infiltration of neutrophils and macrophages in periarticular tissues, suggesting SLPI's role in immune regulation. The authors strengthened their findings by demonstrating a direct interaction between SLPI and B. burgdorferi through BASEHIT library screening and FACS analysis. Further investigation of SLPI as a target could lead to valuable clinical applications.
The conclusions of this paper are mostly well supported by data. And the authors were responsive to the reviewers' comments.
Comments on revised version:
The authors have thoroughly addressed the previous concerns and improved the manuscript. The revisions have strengthened both the conclusions. I have no additional suggestions for improvement and recommend this manuscript for publication.
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Reviewer #2 (Public review):
Summary:
This manuscript by Yu and coworkers investigates the potential role of Secretory leukocyte protease inhibitor (SLPI) in Lyme arthritis. They show that, after needle inoculation of the Lyme disease (LD) agent, B. burgdorferi, compared to wild type mice, a SLPI-deficient mouse suffers elevated bacterial burden, joint swelling and inflammation, pro-inflammatory cytokines in the joint, and levels of serum neutrophil elastase (NE). They suggest that SLPI levels of Lyme disease patients are diminished relative to healthy controls. Finally, they find that SLPI may interact directly the B. burgdorferi.
Strengths:
Many of these observations are interesting and the use of SLPI-deficient mice is useful (and has not previously been done).
Weaknesses:
(a) The known role of SLPI in dampening inflammation and …
Reviewer #2 (Public review):
Summary:
This manuscript by Yu and coworkers investigates the potential role of Secretory leukocyte protease inhibitor (SLPI) in Lyme arthritis. They show that, after needle inoculation of the Lyme disease (LD) agent, B. burgdorferi, compared to wild type mice, a SLPI-deficient mouse suffers elevated bacterial burden, joint swelling and inflammation, pro-inflammatory cytokines in the joint, and levels of serum neutrophil elastase (NE). They suggest that SLPI levels of Lyme disease patients are diminished relative to healthy controls. Finally, they find that SLPI may interact directly the B. burgdorferi.
Strengths:
Many of these observations are interesting and the use of SLPI-deficient mice is useful (and has not previously been done).
Weaknesses:
(a) The known role of SLPI in dampening inflammation and inflammatory damage by inhibition of NE makes the enhanced inflammation in the joint of B. burgdorferi-infected mice a predicted result; (b) The potential contribution of the greater bacterial burden to the enhanced inflammation is acknowledged but not experimentally addressed; (c) The relationship of SLPI binding by B. burgdorferi to the enhanced disease of SLPI-deficient mice is not addressed in this study, making the inclusion of this observation in this manuscript incomplete; and (d) assessment of SLPI levels in healthy controls vs. Lyme disease patients is inadequate.
Comments on revised verson:
Several of the points were addressed in the revised manuscript, but the following issues remain:
Previous point that the relationship of SLPI binding to B. burgdorferi to the enhanced disease of SLPI-deficient mice is not investigated: The authors indicate that such investigations are ongoing. In the absence of any findings, I recommend that their interesting BASEHIT and subsequent studies be presented in a future study, which would have high impact.
Previous recommendation 1: (The authors added lines 267-68, not 287-68). This ambiguity is acknowledged but remains. In addition, in the revised manuscript, the authors state "However, these data also emphasize the importance of SLPI in controlling the development of inflammation in periarticular tissues of B. burgdorferi-infected mice." Given acknowledged limitations of interpretation, "suggest" would be more appropriate than "emphasize".
Previous recommendation 5: The lack of clinical samples can be a challenge. Nevertheless, 4 of the 7 samples from LD patients are from individuals suffering from EM rather than arthritis (i.e., the manifestation that is the topic of the study) and some who are sampled multiple times, make an objective statistical comparison difficult. I don't have a suggestion as to how to address the difference in number of samples from a given subject. However, the authors could consider segregating EM vs. LA in their analysis (although it appears that limiting the comparison between HC and LA patients would not reveal a statistical difference).
Previous recommendation 6: Given that binding of SLPI to the bacterial surface is an essential aspect of the authors' model, and that the ELISA assay to indicate SLPI binding used cell lysates rather than intact bacteria, a control PI staining to validate the integrity of bacteria seems reasonable.
Previous recommendation 8: The inclusion of a no serum control (that presumably shows 100% viability) would validate the authors' assertion that 20% serum has bactericidal activity.
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Reviewer #3 (Public review):
Summary:
The authors investigated the role of secretory leukocyte protease inhibitors (SLPI) in developing Lyme disease in mice infected with Borrelia burgdorferi. Using a combination of histological, gene expression, and flow cytometry analyses, they demonstrated significantly higher bacterial burden and elevated neutrophil and macrophage infiltration in SLPI-deficient mouse ankle joints. Furthermore, they also showed direct interaction of SLPI with B. burgdorferi, which likely depletes the local environment of SLPI and causes excessive protease activity. These results overall suggest ankle tissue inflammation in B. burgdorferi-infected mice is driven by unchecked protease activity.
Strengths:
Utilizing a comprehensive suite of techniques, this is the first study showing the importance of …
Reviewer #3 (Public review):
Summary:
The authors investigated the role of secretory leukocyte protease inhibitors (SLPI) in developing Lyme disease in mice infected with Borrelia burgdorferi. Using a combination of histological, gene expression, and flow cytometry analyses, they demonstrated significantly higher bacterial burden and elevated neutrophil and macrophage infiltration in SLPI-deficient mouse ankle joints. Furthermore, they also showed direct interaction of SLPI with B. burgdorferi, which likely depletes the local environment of SLPI and causes excessive protease activity. These results overall suggest ankle tissue inflammation in B. burgdorferi-infected mice is driven by unchecked protease activity.
Strengths:
Utilizing a comprehensive suite of techniques, this is the first study showing the importance of anti-protease-protease balance in the development of periarticular joint inflammation in Lyme disease.
Weaknesses:
Due to the limited sample availability, the authors investigated the serum level of SLPI in both Lyme arthritis patients and patients with earlier disease manifestations. This limitation is thoroughly discussed in the manuscript.
Comments on revised version:
I thank the authors for considering my comments carefully.
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Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
Summary:
This study demonstrates the significant role of secretory leukocyte protease inhibitor (SLPI) in regulating B. burgdorferi-induced periarticular inflammation in mice. They found that SLPI-deficient mice showed significantly higher B. burgdorferi infection burden in ankle joints compared to wild-type controls. This increased infection was accompanied by infiltration of neutrophils and macrophages in periarticular tissues, suggesting SLPI's role in immune regulation. The authors strengthened their findings by demonstrating a direct interaction between SLPI and B. burgdorferi through BASEHIT library screening and FACS analysis. Further investigation of SLPI as a target could lead to valuable clinical applications.
The …
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
Summary:
This study demonstrates the significant role of secretory leukocyte protease inhibitor (SLPI) in regulating B. burgdorferi-induced periarticular inflammation in mice. They found that SLPI-deficient mice showed significantly higher B. burgdorferi infection burden in ankle joints compared to wild-type controls. This increased infection was accompanied by infiltration of neutrophils and macrophages in periarticular tissues, suggesting SLPI's role in immune regulation. The authors strengthened their findings by demonstrating a direct interaction between SLPI and B. burgdorferi through BASEHIT library screening and FACS analysis. Further investigation of SLPI as a target could lead to valuable clinical applications.
The conclusions of this paper are mostly well supported by data, but two aspects need attention:
(1) Cytokine Analysis:
The serum cytokine/chemokine profile analysis appears without TNF-alpha data. Given TNF-alpha's established role in inflammatory responses, comparing its levels between wild-type and infected B. burgdorferi conditions would provide valuable insight into the inflammatory mechanism.
(2) Sample Size Concerns:
While the authors note limitations in obtaining Lyme disease patient samples, the control group is notably smaller than the patient group. This imbalance should either be addressed by including additional healthy controls or explicitly justified in the methodology section.
We thank the reviewer for the careful review and positive comments.
(1) We did look into the level of TNF-alpha in both WT and SLPI-/- mice with and without B. burgdorferi infection. At serum level, using ELISA, we did not observe any significant difference between all four groups. At gene expression level, using RT-qPCR on the tibiotarsal tissue, we also did not observe any significant differences. Our RT-qPCR result is consistent with the previous microarray study using the whole murine joint tissue (DOI: 10.4049/jimmunol.177.11.7930). The microarray study did not show significant changes in TNF-alpha level in C57BL/6 mice following B. burgdorferi infection. A brief discussion has been added, and the above data is provided as Supplemental figure 4 in the revised manuscript, line 334-339, and 756-763.
(2) We agree with the reviewer that the control group is smaller than the patient group. Among the archived samples that are available, the number of adult healthy controls are limited. It has been shown that the serum level of SLPI in healthy volunteers is in average about 40 ng/ml (DOI: 10.3389/fimmu.2019.00664 and 10.1097/00003246-200005000-00003). The median level in the healthy control in our data was 38.92 ng/ml, which is comparable to the previous results. A brief discussion has been added in the revised manuscript, line 364-369.
Reviewer #2 (Public review):
Summary:
This manuscript by Yu and coworkers investigates the potential role of Secretory leukocyte protease inhibitor (SLPI) in Lyme arthritis. They show that, after needle inoculation of the Lyme disease (LD) agent, B. burgdorferi, compared to wild type mice, a SLPI-deficient mouse suffers elevated bacterial burden, joint swelling and inflammation, pro-inflammatory cytokines in the joint, and levels of serum neutrophil elastase (NE). They suggest that SLPI levels of Lyme disease patients are diminished relative to healthy controls. Finally, they find that SLPI may interact directly the B. burgdorferi.
Strengths:
Many of these observations are interesting and the use of SLPI-deficient mice is useful (and has not previously been done).
We appreciate the reviewer’s careful reading and positive comments.
Weaknesses:
(a) The known role of SLPI in dampening inflammation and inflammatory damage by inhibition of NE makes the enhanced inflammation in the joint of B. burgdorferi-infected mice a predicted result;
We agree that the observation of the elevated NE level and the enhanced inflammation is theoretically likely. Indeed, that was the hypothesis that we explored, and often what is theoretically possible does not turn out to occur. In addition, despite the known contribution of neutrophils to the severity of murine Lyme arthritis, the importance of the neutrophil serine proteases and anti-protease has not been specifically studied, and neutrophils secrete many factors. Therefore, our data fill an important gap in the knowledge of murine Lyme arthritis development – and set the stage for the further exploration of this hypothesis in the genesis of human Lyme arthritis.
(b) The potential contribution of the greater bacterial burden to the enhanced inflammation is not addressed;
We agree with the reviewer’s viewpoint that the increased infection burden in the tibiotarsal tissue of the infected SLPI-/- mice could contribute to the enhanced inflammation. A brief discussion of this possibility has been added in the revised manuscript, line 287-288.
(c) The relationship of SLPI binding by B. burgdorferi to the enhanced disease of SLPI-deficient mice is not clear; and
We agree with the reviewer that we have not shown the importance of the SLPI-B. burgdorferi binding in the development of periarticular inflammation. It is an ongoing project in our lab to identify the SLPI binding partner in B. burgdorferi. Our hypothesis is that SLPI could bind and inhibit an unknown B. burgdorferi virulence factor that contributes to murine Lyme arthritis. A brief discussion has been added in the revised manuscript, line 401-407.
(d) Several methodological aspects of the study are unclear.
We appreciate the critique. We have modified the methods section in greater detail in the revised manuscript.
Reviewer #3 (Public review):
Summary:
The authors investigated the role of secretory leukocyte protease inhibitors (SLPI) in developing Lyme disease in mice infected with Borrelia burgdorferi. Using a combination of histological, gene expression, and flow cytometry analyses, they demonstrated significantly higher bacterial burden and elevated neutrophil and macrophage infiltration in SLPI-deficient mouse ankle joints. Furthermore, they also showed direct interaction of SLPI with B. burgdorferi, which likely depletes the local environment of SLPI and causes excessive protease activity. These results overall suggest ankle tissue inflammation in B. burgdorferi-infected mice is driven by unchecked protease activity.
Strengths:
Utilizing a comprehensive suite of techniques, this is the first study showing the importance of anti-protease-protease balance in the development of periarticular joint inflammation in Lyme disease.
We greatly appreciate the reviewer’s careful reading and positive comments.
Weaknesses:
Due to the limited sample availability, the authors investigated the serum level of SLPI in both in Lyme arthritis patients and patients with earlier disease manifestations.
We agree with the reviewer that it would be ideal to have more samples from Lyme arthritis patients. However, among the available archived samples, samples from Lyme arthritis patients are limited. For the samples from patients with single EM, the symptom persisted into 3-4 month after diagnosis, the same timeframe when acute arthritis is developed. A brief discussion has been added in the revised manuscript, line 364-369.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
(1) In Figure 2, for histological scoring, do they have similar n numbers?
In panel B, 20 infected WT mice and 19 infected SLPI-/- mice were examined. In panel D, 13 infected WT and SLPI-/- mice were examined. Without infection, WT and SLPI-/- mice do not develop spontaneous arthritis. Due to the slow breeding of the SLPI-/- mice, a small number of uninfected control animals were used. All the supporting data values are provided in the supplemental excel.
(2) In Figure 3, for macrophage population analysis, maybe consider implementing Ly6G-negative gating strategy to prevent neutrophil contamination in macrophage population?
We appreciate reviewer’s suggestion. We have analyzed the data using the Ly6G-negative gating strategy and provided the result in the Supplemental figure 1. The two gating strategies showed consistent result, significantly higher percentage of infiltrating macrophages in the tibiotarsal tissue from infected SLPI-/- mice, line 154-158, line 726-729.
Reviewer #2 (Recommendations for the authors):
(1) The investigators should address the possibility that much of the enhanced inflammatory features of infected SLPI-deficient mice are simply due to the higher bacterial load in the joint.
We agree with the reviewer’s viewpoint that the increased infection burden in the tibiotarsal tissue of the infected SLPI-/- mice could contribute to the enhanced inflammation. A brief discussion of this possibility has been added in the revised manuscript, line 287-288.
(2) Fig. 1. (A) There is no statistically significant difference in the bacterial load in the heart or skin, in contrast to the tibiotarsal joint. It would be of interest to know whether other tissues that are routinely sampled to assess the bacterial load, such as injection site, knee, and bladder, also harbored increased bacterial load in SLPI-deficient mice. (B) Heart and joint burden were measured at "21-28" days. The two time points should be analyzed separately rather than pooled.
(A) We appreciate the reviewer’s suggestion. We agree that looking into the infection load in other tissues is helpful. However, studies into murine Lyme arthritis have been predominantly focused on tibiotarsal tissue, which displays the most consistent and prominent swelling that’s easy to observe and measure. Thus, we focused on the tibiotarsal joint in our study. (B) We collected the heart and joint tissue approximately 3-week post infection within a 3-day window based on the feasibility and logistics of the laboratory. Using “21-28 d”, we meant to describe between 21 to 24 days post infection. We apologize for the mislabeling and it has been corrected it in the revised manuscript. In the methods, we defined the timeframe as “Mice were euthanized approximately 3-week post infection within a 3-day window (between 21 to 24 dpi) based on the feasibility and logistics of the laboratory”, line 464-466. In the results and figure legend, we corrected it as “between 21 to 24 dpi”.
(3) Fig. 2. (A) The same ambiguity as to the days post-infection as cited above in Point 2B exists in this figure. (B) Panel B: Caliper measurements to assess joint swelling should be utilized rather than visual scoring. (In addition, the legend should make clear that the black circles represent mock-infected mice.)
(A) The histology scoring, and histopathology examination were performed at the same time as heart and joint tissue collection, approximately 3 weeks post infection within a 3-day window based on the feasibility and logistics of the laboratory. We apologize for the mislabeling and it has been corrected in the revised manuscript. (B) We appreciate the reviewer’s suggestion. However, our extensive experience is that caliper measurement can alter the assessment of swelling by placing pressure on the joints and did not produce consistent results. Double blinded scoring was thus performed. Histopathology examination was performed by an independent pathologist and confirmed the histology score and provided additional measurements.
(4) Fig. 3. (A) See Point 2B. (B) For Panels C-E, uninfected controls are lacking.
We apologize for this omission. Uninfected controls have been provided in Figure 3 in the revised manuscript.
(5) Fig. 4. Fig. 4. Some LD subjects were sampled multiple times (5 samples from 3 subjects with Lyme arthritis; 13 samples from 4 subjects with EM), and samples from same individuals apparently are treated as biological replicates in the statistical analysis. In contrast, the 5 healthy controls were each sampled only once.
We agree with the reviewer that the control group is smaller than the patient group. Among the archived samples that are available, the number of adult healthy controls are limited, and sampled once. We used these samples to establish the baseline level of SLPI in the serum. It has been shown that the serum level of SLPI in healthy volunteers is in average about 40 ng/ml (DOI: 10.3389/fimmu.2019.00664 and 10.1097/00003246-200005000-00003). The median level in the healthy control in our data was 38.92 ng/ml, which is comparable to the previous results. A brief discussion has been added in the revised manuscript, line 364-369.
(6) Fig. 5. (A) Panel A: does binding occur when intact bacteria are used? (B) Panels B, C: Were bacteria probed with PI to indicate binding likely to occur to surface? How many biological replicates were performed for each panel? Is "antibody control" a no SLPI control? What is the blue line?
Actively growing B. burgdorferi were collected and used for binding assays. We do not permeabilize the bacteria for flow cytometry. Thus, all the binding detected occurs to the bacterial surface. Three biological replicates were performed for each panel. The antibody control is no SLPI control. For panel D, the bacteria were stained with Hoechst, which shows the morphology of bacteria. We apologize for the missing information. A complete and detailed description of Figure 5 has been provided in both methods and figure legend in the revised manuscript.
(7) Sup Fig. 1. (A) Panel A: Was this experiment performed multiple times? I.e., how many biological replicates? (B) Panel B: Strain should be specified.
The binding assay to B. burgdorferi B31A was performed two times. In panel B, B. burgdorferi B31A3 was used. We apologize for the missing information. A complete and detailed description has been provided in the figure legend in the revised manuscript.
(8) Fig. S2. It is not clear that the condition (20% serum) has any bactericidal activity, so the potential protective activity of SLPI cannot be determined. (Typical serum killing assays in the absence of specific antibody utilized 40% serum.)
In Fig. S2, panel B, the first two bars (without SLPI, with 20% WT anti serum) showed around 40% viability. It indicates that the 20% WT anti serum has bactericidal activity. Serum was collected from B. burgdorferi-infected WT mice at 21 dpi, which should contain polyclonal antibody against B. burgdorferi.
Reviewer #3 (Recommendations for the authors):
It was a pleasure to review! I congratulate the authors on this elegant study. I think the manuscript is very well-written and clearly conveys the research outcomes. I only have minor suggestions to improve the readability of the text.
We greatly appreciate the reviewer’s recognition of our work.
Line 92: Please briefly summarize the key results of the study at the end of the introduction section.
We appreciate the reviewer’s suggestion. A brief summary has been added in the revised manuscript, line 93-103.
Line 108: Why is the inflammation significantly occurred only in ankle joints of SLPI-I mice? Could you please provide a brief explanation?
The inflammation may also happen in other joints the B. burgdorferi infected SLPI-/- mice, which has not been studied. The study into murine Lyme arthritis has been predominantly done in the tibiotarsal tissue, which displays the most prominent swelling that’s easy to observe and measure. Thus, we focused on the tibiotarsal joint in our study.
Line 136: Please also include the gene names in Figure 3.
We apologize for the omission. Gene names has been included in figure legend in the revised manuscript.
Line 181: Please briefly introduce BASEHIT. Why did you use this tool? What are the benefits?
We appreciate the reviewer’s suggestion. We have provided a brief introduction on BASEHIT in the revised manuscript, line 216-218.
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eLife Assessment
This study presents a valuable finding on the role of secretory leukocyte protease inhibitors (SLPI) in developing Lyme disease in mice infected with Borrelia burgdorferi. The evidence supporting the claims of the authors is solid, although there are a few concerns that need to be addressed, including patient sample sizes, and the potential contribution of the greater bacterial burden to the enhanced inflammation. This paper would be of interest to scientists in the infectious inflammatory disease field.
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Reviewer #1 (Public review):
Summary:
This study demonstrates the significant role of secretory leukocyte protease inhibitor (SLPI) in regulating B. burgdorferi-induced periarticular inflammation in mice. They found that SLPI-deficient mice showed significantly higher B. burgdorferi infection burden in ankle joints compared to wild-type controls. This increased infection was accompanied by infiltration of neutrophils and macrophages in periarticular tissues, suggesting SLPI's role in immune regulation. The authors strengthened their findings by demonstrating a direct interaction between SLPI and B. burgdorferi through BASEHIT library screening and FACS analysis. Further investigation of SLPI as a target could lead to valuable clinical applications.
The conclusions of this paper are mostly well supported by data, but two aspects need …
Reviewer #1 (Public review):
Summary:
This study demonstrates the significant role of secretory leukocyte protease inhibitor (SLPI) in regulating B. burgdorferi-induced periarticular inflammation in mice. They found that SLPI-deficient mice showed significantly higher B. burgdorferi infection burden in ankle joints compared to wild-type controls. This increased infection was accompanied by infiltration of neutrophils and macrophages in periarticular tissues, suggesting SLPI's role in immune regulation. The authors strengthened their findings by demonstrating a direct interaction between SLPI and B. burgdorferi through BASEHIT library screening and FACS analysis. Further investigation of SLPI as a target could lead to valuable clinical applications.
The conclusions of this paper are mostly well supported by data, but two aspects need attention:
(1) Cytokine Analysis:
The serum cytokine/chemokine profile analysis appears without TNF-alpha data. Given TNF-alpha's established role in inflammatory responses, comparing its levels between wild-type and infected B. burgdorferi conditions would provide valuable insight into the inflammatory mechanism.
(2) Sample Size Concerns:
While the authors note limitations in obtaining Lyme disease patient samples, the control group is notably smaller than the patient group. This imbalance should either be addressed by including additional healthy controls or explicitly justified in the methodology section. -
Reviewer #2 (Public review):
Summary:
This manuscript by Yu and coworkers investigates the potential role of Secretory leukocyte protease inhibitor (SLPI) in Lyme arthritis. They show that, after needle inoculation of the Lyme disease (LD) agent, B. burgdorferi, compared to wild type mice, a SLPI-deficient mouse suffers elevated bacterial burden, joint swelling and inflammation, pro-inflammatory cytokines in the joint, and levels of serum neutrophil elastase (NE). They suggest that SLPI levels of Lyme disease patients are diminished relative to healthy controls. Finally, they find that SLPI may interact directly the B. burgdorferi.
Strengths:
Many of these observations are interesting and the use of SLPI-deficient mice is useful (and has not previously been done).
Weaknesses:
(a) The known role of SLPI in dampening inflammation and …
Reviewer #2 (Public review):
Summary:
This manuscript by Yu and coworkers investigates the potential role of Secretory leukocyte protease inhibitor (SLPI) in Lyme arthritis. They show that, after needle inoculation of the Lyme disease (LD) agent, B. burgdorferi, compared to wild type mice, a SLPI-deficient mouse suffers elevated bacterial burden, joint swelling and inflammation, pro-inflammatory cytokines in the joint, and levels of serum neutrophil elastase (NE). They suggest that SLPI levels of Lyme disease patients are diminished relative to healthy controls. Finally, they find that SLPI may interact directly the B. burgdorferi.
Strengths:
Many of these observations are interesting and the use of SLPI-deficient mice is useful (and has not previously been done).
Weaknesses:
(a) The known role of SLPI in dampening inflammation and inflammatory damage by inhibition of NE makes the enhanced inflammation in the joint of B. burgdorferi-infected mice a predicted result; (b) The potential contribution of the greater bacterial burden to the enhanced inflammation is not addressed; (c) The relationship of SLPI binding by B. burgdorferi to the enhanced disease of SLPI-deficient mice is not clear; and (d) Several methodological aspects of the study are unclear.
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Reviewer #3 (Public review):
Summary:
The authors investigated the role of secretory leukocyte protease inhibitors (SLPI) in developing Lyme disease in mice infected with Borrelia burgdorferi. Using a combination of histological, gene expression, and flow cytometry analyses, they demonstrated significantly higher bacterial burden and elevated neutrophil and macrophage infiltration in SLPI-deficient mouse ankle joints. Furthermore, they also showed direct interaction of SLPI with B. burgdorferi, which likely depletes the local environment of SLPI and causes excessive protease activity. These results overall suggest ankle tissue inflammation in B. burgdorferi-infected mice is driven by unchecked protease activity.
Strengths:
Utilizing a comprehensive suite of techniques, this is the first study showing the importance of …
Reviewer #3 (Public review):
Summary:
The authors investigated the role of secretory leukocyte protease inhibitors (SLPI) in developing Lyme disease in mice infected with Borrelia burgdorferi. Using a combination of histological, gene expression, and flow cytometry analyses, they demonstrated significantly higher bacterial burden and elevated neutrophil and macrophage infiltration in SLPI-deficient mouse ankle joints. Furthermore, they also showed direct interaction of SLPI with B. burgdorferi, which likely depletes the local environment of SLPI and causes excessive protease activity. These results overall suggest ankle tissue inflammation in B. burgdorferi-infected mice is driven by unchecked protease activity.
Strengths:
Utilizing a comprehensive suite of techniques, this is the first study showing the importance of anti-protease-protease balance in the development of periarticular joint inflammation in Lyme disease.
Weaknesses:
Due to the limited sample availability, the authors investigated the serum level of SLPI in both in Lyme arthritis patients and patients with earlier disease manifestations.
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Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
This study demonstrates the significant role of secretory leukocyte protease inhibitor (SLPI) in regulating B. burgdorferi-induced periarticular inflammation in mice. They found that SLPI-deficient mice showed significantly higher B. burgdorferi infection burden in ankle joints compared to wild-type controls. This increased infection was accompanied by infiltration of neutrophils and macrophages in periarticular tissues, suggesting SLPI's role in immune regulation. The authors strengthened their findings by demonstrating a direct interaction between SLPI and B. burgdorferi through BASEHIT library screening and FACS analysis. Further investigation of SLPI as a target could lead to valuable clinical applications.
The conclusions of this paper are mostly well supported …
Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
This study demonstrates the significant role of secretory leukocyte protease inhibitor (SLPI) in regulating B. burgdorferi-induced periarticular inflammation in mice. They found that SLPI-deficient mice showed significantly higher B. burgdorferi infection burden in ankle joints compared to wild-type controls. This increased infection was accompanied by infiltration of neutrophils and macrophages in periarticular tissues, suggesting SLPI's role in immune regulation. The authors strengthened their findings by demonstrating a direct interaction between SLPI and B. burgdorferi through BASEHIT library screening and FACS analysis. Further investigation of SLPI as a target could lead to valuable clinical applications.
The conclusions of this paper are mostly well supported by data, but two aspects need attention:
(1) Cytokine Analysis:
The serum cytokine/chemokine profile analysis appears without TNF-alpha data. Given TNF-alpha's established role in inflammatory responses, comparing its levels between wild-type and infected B. burgdorferi conditions would provide valuable insight into the inflammatory mechanism.
(2) Sample Size Concerns:
While the authors note limitations in obtaining Lyme disease patient samples, the control group is notably smaller than the patient group. This imbalance should either be addressed by including additional healthy controls or explicitly justified in the methodology section.
We thank the reviewer for the careful review and positive comments.
(1) We did look into the level of TNF-alpha in both WT and SLPI-/- mice with and without B. burgdorferi infection. At serum level, using ELISA, we did not observe any significant difference between all four groups. At gene expression level, using RT-qPCR on the tibiotarsal tissue, we also did not observe any significant differences. Our RT-qPCR result is consistent with the previous microarray study using the whole murine joint tissue (DOI: 10.4049/jimmunol.177.11.7930). The microarray study did not show significant changes in TNF-alpha level in C57BL/6 mice following B. burgdorferi infection. The above data suggest that TNF-alpha does not involve in SLPI-regulated immune responses in the murine tibiotarsal tissue following B. burgdorferi infection. A brief discussion will be added, and the above data will be provided as a supplemental figure in the revised manuscript.
(2) We agree with the reviewer that the control group is smaller than the patient group. Among the archived samples that are available, the number of adult healthy controls are limited. It has been shown that the serum level of SLPI in healthy volunteers is in average about 40 ng/ml (DOI: 10.3389/fimmu.2019.00664 and 10.1097/00003246-200005000-00003). The median level in the healthy control in our data was 38.92 ng/ml, which is comparable to the previous results. A brief discussion will be added in the revised manuscript.
Reviewer #2 (Public review):
Summary:
This manuscript by Yu and coworkers investigates the potential role of Secretory leukocyte protease inhibitor (SLPI) in Lyme arthritis. They show that, after needle inoculation of the Lyme disease (LD) agent, B. burgdorferi, compared to wild type mice, a SLPI-deficient mouse suffers elevated bacterial burden, joint swelling and inflammation, pro-inflammatory cytokines in the joint, and levels of serum neutrophil elastase (NE). They suggest that SLPI levels of Lyme disease patients are diminished relative to healthy controls. Finally, they find that SLPI may interact directly the B. burgdorferi.
Strengths:
Many of these observations are interesting and the use of SLPI-deficient mice is useful (and has not previously been done).
We appreciate the reviewer’s careful reading and positive comments.
Weaknesses:
(a) The known role of SLPI in dampening inflammation and inflammatory damage by inhibition of NE makes the enhanced inflammation in the joint of B. burgdorferi-infected mice a predicted result;
We agree that the observation of the elevated NE level and the enhanced inflammation is theoretically likely. Indeed, that was the hypothesis that we explored, and often what is theoretically possible does not turn out to occur. In addition, despite the known contribution of neutrophils to the severity of murine Lyme arthritis, the importance of the neutrophil serine proteases and anti-protease has not been specifically studied, and neutrophils secrete many factors. Therefore, our data fill an important gap in the knowledge of murine Lyme arthritis development – and set the stage for the further exploration of this hypothesis in the genesis of human Lyme arthritis.
(b) The potential contribution of the greater bacterial burden to the enhanced inflammation is not addressed;
We agree with the reviewer’s viewpoint that the increased infection burden in the tibiotarsal tissue of the infected SLPI-/- mice could contribute to the enhanced inflammation. A brief discussion of this possibility will be added to the revised manuscript.
(c) The relationship of SLPI binding by B. burgdorferi to the enhanced disease of SLPI-deficient mice is not clear; and
We agree with the reviewer that we have not shown the importance of the SLPI-B. burgdorferi binding in the development of periarticular inflammation. It is an ongoing project in our lab to identify the SLPI binding partner in B. burgdorferi. Our hypothesis is that SLPI could bind and inhibit an unknown B. burgdorferi virulence factor that contributes to murine Lyme arthritis. We will include the above discussion in the revised manuscript.
(d) Several methodological aspects of the study are unclear.
We appreciate the critique and will modify the method session in greater detail in the revised manuscript.
Reviewer #3 (Public review):
Summary:
The authors investigated the role of secretory leukocyte protease inhibitors (SLPI) in developing Lyme disease in mice infected with Borrelia burgdorferi. Using a combination of histological, gene expression, and flow cytometry analyses, they demonstrated significantly higher bacterial burden and elevated neutrophil and macrophage infiltration in SLPI-deficient mouse ankle joints. Furthermore, they also showed direct interaction of SLPI with B. burgdorferi, which likely depletes the local environment of SLPI and causes excessive protease activity. These results overall suggest ankle tissue inflammation in B. burgdorferi-infected mice is driven by unchecked protease activity.
Strengths:
Utilizing a comprehensive suite of techniques, this is the first study showing the importance of anti-protease-protease balance in the development of periarticular joint inflammation in Lyme disease.
We greatly appreciate the reviewer’s careful reading and positive comments.
Weaknesses:
Due to the limited sample availability, the authors investigated the serum level of SLPI in both in Lyme arthritis patients and patients with earlier disease manifestations.
We agree with the reviewer that it would be ideal to have more samples from Lyme arthritis patients. However, among the available archived samples, samples from Lyme arthritis patients are limited. For the samples from patients with single EM, the symptom persisted into 3-4 month after diagnosis, the same timeframe when arthritis is developed. We will add the above discussion in the revised manuscript.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
(1) In Figure 2, for histological scoring, do they have similar n numbers?
In panel B, 20 infected WT mice and 19 infected SLPI-/- mice were examined. In panel D, 13 infected WT and SLPI-/- mice were examined. Without infection, WT and SLPI-/- mice do not develop spontaneous arthritis. Due to the slow breeding of the SLPI-/- mice, a small number of uninfected control animals were used.
(2) In Figure 3, for macrophage population analysis, maybe consider implementing Ly6G-negative gating strategy to prevent neutrophil contamination in macrophage population?
We appreciate reviewer’s suggestion. We will analyze the data using the Ly6G-negative gating strategy and provide the result in a supplemental figure. We will compare the results using the two gating strategies in the revised manuscript.
Reviewer #2 (Recommendations for the authors):
(1) The investigators should address the possibility that much of the enhanced inflammatory features of infected SLPI-deficient mice are simply due to the higher bacterial load in the joint.
We agree with the reviewer’s viewpoint that the increased infection burden in the tibiotarsal tissue of the infected SLPI-/- mice could contribute to the enhanced inflammation. A brief discussion of this possibility will be added to the revised manuscript.
(2) Fig. 1. (A) There is no statistically significant difference in the bacterial load in the heart or skin, in contrast to the tibiotarsal joint. It would be of interest to know whether other tissues that are routinely sampled to assess the bacterial load, such as injection site, knee, and bladder, also harbored increased bacterial load in SLPI-deficient mice. (B) Heart and joint burden were measured at "21-28" days. The two time points should be analyzed separately rather than pooled.
(A) We appreciate the reviewer’s suggestion. We agree that looking into the infection load in other tissues is helpful. However, studies into murine Lyme arthritis have been predominantly focused on tibiotarsal tissue, which displays the most consistent and prominent swelling that’s easy to observe and measure. Thus, we focused on the tibiotarsal joint in our study. (B) We collected the heart and joint tissue approximately 3-week post infection within a 3-day window based on the feasibility and logistics of the laboratory. Using “21-28 d”, we meant to describe between 21-24 days post infection. We apologize for the mislabeling and will correct it in the revised manuscript, stating approximately 3 weeks in the results, and defining approximately 3-weeks as between 21-24 days in the methods.
(3) Fig. 2. (A) The same ambiguity as to the days post-infection as cited above in Point 2B exists in this figure. (B) Panel B: Caliper measurements to assess joint swelling should be utilized rather than visual scoring. (In addition, the legend should make clear that the black circles represent mock-infected mice.)
(A) The histology scoring, and histopathology examination were performed at the same time as heart and joint tissue collection, approximately 3 weeks post infection within a 3-day window based on the feasibility and logistics of the laboratory. We apologize for the mislabeling and will correct it in the revised manuscript. (B) We appreciate the reviewer’s suggestion. However, our extensive experience is that caliper measurement can alter the assessment of swelling by placing pressure on the joints and did not produce consistent results. Double blinded scoring was thus performed. Histopathology examination was performed by an independent pathologist and confirmed the histology score and provided additional measurements.
(4) Fig. 3. (A) See Point 2B. (B) For Panels C-E, uninfected controls are lacking.
We apologize for this omission. Uninfected controls will be provided in the revised manuscript.
(5) Fig. 4. Fig. 4. Some LD subjects were sampled multiple times (5 samples from 3 subjects with Lyme arthritis; 13 samples from 4 subjects with EM), and samples from same individuals apparently are treated as biological replicates in the statistical analysis. In contrast, the 5 healthy controls were each sampled only once.
We agree with the reviewer that the control group is smaller than the patient group. Among the archived samples that are available, the number of adult healthy controls are limited, and sampled once. We used these samples to establish the baseline level of SLPI in the serum. It has been shown that the serum level of SLPI in healthy volunteers is in average about 40 ng/ml (DOI: 10.3389/fimmu.2019.00664 and 10.1097/00003246-200005000-00003). The median level in the healthy control in our data was 38.92 ng/ml, which is comparable to the previous results. A brief discussion will be added in the revised manuscript.
(6) Fig. 5. (A) Panel A: does binding occur when intact bacteria are used? (B) Panels B, C: Were bacteria probed with PI to indicate binding likely to occur to surface? How many biological replicates were performed for each panel? Is "antibody control" a no SLPI control? What is the blue line?
Actively growing B. burgdorferi were collected and used for binding assays. We do not permeabilize the bacteria for flow cytometry. Thus, all the binding detected occurs to the bacterial surface. Three biological replicates were performed for each panel. The antibody control is no SLPI control. For panel D, the bacteria were stained with Hoechst, which shows the morphology of bacteria. We apologize for the missing information. A complete and detailed description of Figure 5 will be provided in the revised manuscript.
(7) Sup Fig. 1. (A) Panel A: Was this experiment performed multiple times? I.e., how many biological replicates? (B) Panel B: Strain should be specified.
The binding assay to B. burgdorferi B31A was performed two times. In panel B, B. burgdorferi B31A3 was used. We apologize for the missing information. A complete and detailed description will be provided in the revised manuscript.
(8) Fig. S2. It is not clear that the condition (20% serum) has any bactericidal activity, so the potential protective activity of SLPI cannot be determined. (Typical serum killing assays in the absence of specific antibody utilized 40% serum.)
In Fig. S2, panel B, the first two bars (without SLPI, with 20% WT anti serum) showed around 40% viability. It indicates that the 20% WT anti serum has bactericidal activity. Serum was collected from B. burgdorferi-infected WT mice at 21 dpi, which should contain polyclonal antibody against B. burgdorferi.
Reviewer #3 (Recommendations for the authors):
It was a pleasure to review! I congratulate the authors on this elegant study. I think the manuscript is very well-written and clearly conveys the research outcomes. I only have minor suggestions to improve the readability of the text.
We greatly appreciate the reviewer’s recognition of our work.
Line 92: Please briefly summarize the key results of the study at the end of the introduction section.
We appreciate the reviewer’s suggestion. A brief summary will be added in the revised manuscript.
Line 108: Why is the inflammation significantly occurred only in ankle joints of SLPI-I mice? Could you please provide a brief explanation?
The inflammation may also happen in other joints the B. burgdorferi infected SLPI-/- mice, which has not been studied. The study into murine Lyme arthritis has been predominantly done in the tibiotarsal tissue, which displays the most prominent swelling that’s easy to observe and measure. Thus, we focused on the tibiotarsal joint in our study.
Line 136: Please also include the gene names in Figure 3.
We apologize for the omission. Gene names will be included in the revised manuscript.
Line 181: Please briefly introduce BASEHIT. Why did you use this tool? What are the benefits?
We appreciate the reviewer’s suggestion. We will provide more background information on BASEHIT in the revised manuscript.
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