VEGFR Blockade Reduces Mycobacterium tuberculosis-Induced Lung Pathology in Immunocompromised Mice

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled “VEGFR Blockade Reduces Mycobacterium tuberculosis-Induced Lung Pathology in Immunocompromised Mice” by Herbath et al. examines the effects of the VEGFR1/2 inhibitor SU5416 on lung pathology in H37Rv-infected immunocompromised RAG1KO mice. The authors report that SU5416 treatment significantly reduced monocyte and NK cell infiltration while increasing neutrophil granulocytes in the lungs of infected mice, without altering pulmonary bacterial burden as measured by CFU. Based on these findings, they propose that VEGFR blockade could serve as an adjunctive therapy to standard anti-tuberculosis regimens in immuno-compromised populations. While the study extends the authors’ prior work in immunocompetent Mtb-infected mice, the current manuscript provides limited novelty and lacks mechanistic depth.

 

Major concerns:

1. The study does not include mechanistic experiments to explain how VEGFR blockade alters immune cell recruitment or why bacterial load remains unchanged despite altered cellular infiltration.
2. Mice ranging from 8 to 28 weeks of age were used, with small experimental groups (3–6 mice per group), including both sexes. Age and sex significantly influence lung pathology, granuloma formation, and immune responses in Mtb infection. The data presented (e.g., Fig. 1) do not clarify whether animals were age- and sex-matched within experiments, and pooling data from three separate experiments introduces variability that limits interpretability.
3. The primary experiments appear to use an acute infection model, which does not fully represent chronic adaptive immune responses, despite claims regarding modulation of host immunity.
4. Lung pathology assessment is based on lesion area measured from H&E staining of a single lung lobe, which may not comprehensively reflect total pulmonary disease burden.
5. The authors equate reduced lesion area with reduced inflammation; however, immune cell infiltration alone does not define inflammatory status. No cytokines, chemokines, or other inflammatory mediators were measured to explain the unchanged bacterial load or altered immune composition.
6. Figures 3G and 4 include small sample sizes (n=3–4 mice), but the sex distribution of these animals is not specified.
7. In the chronic infection model (15-week-old B6.C3H-sst1 mice, N=4/group, both sexes, infected for 16 weeks), SU5416 modestly increased neutrophils (p=0.05) without affecting lesion size. Given known sex-based differences in granuloma formation, these results require stratified analysis and discussion.
8. The manuscript does not adequately address limitations, including small sample sizes, mixed age groups, sex effects, and lack of mechanistic insight.

 

Overall, while the topic is relevant and the therapeutic concept is potentially interesting, the study would benefit from clearer experimental design, sex- and age-stratified analyses, inclusion of inflammatory mediator profiling, and mechanistic validation to strengthen its impact and translational significance.

 

Author Response

Response to Reviewer 1.

We sincerely thank Reviewer 1 for their thorough and constructive evaluation of our manuscript. Their comments have helped us identify areas where additional clarification and discussion are needed. We address each concern in detail below.

Response to Major Concerns

 

Major Concern 1

"The study does not include mechanistic experiments to explain how VEGFR blockade alters immune cell recruitment or why bacterial load remains unchanged despite altered cellular infiltration."

Response to Major Concern 1

We appreciate this important observation. We acknowledge that the current study is primarily descriptive/exploratory in nature and does not include dedicated mechanistic experiments. However, we would like to highlight that:

 

•   In previous studies from our laboratory, we extensively analyzed the cytokine profiles of granuloma-associated immune cells. We demonstrated that in immunocompetent mice, despite decreased inflammation, the cellular composition and proportion of cell types within the granuloma remained remarkably similar to control groups following anti-VEGFR1/2 treatment. The activation of T cells, measured by LFA-1 expression and production of interferon (IFN)- γ, was also unchanged despite VEGF-A inhibition. Although SU5416-treated mice had altered expression of several key cytokines including CCL2, CCL4, CCL7, CCL12, CCL22, CXCL6, XCL1, interleukin (IL)-1β, IL-2, IL-6, and IL-10, tumor necrosis factor alpha (TNF- α) and IL-1α but these changes were not significant. Based on this data, the changing cytokine/chemokine profile opened more questions than explained improved lung pathology (Harding et al., Cell Rep. 2019 May 14;27(7):2119-2131.e6. doi: 10.1016/j.celrep.2019.04.072.).

The role of VEGFA–VEGFR1 (FLT1) signaling in monocyte chemoattraction is well-established in the literature (Clauss et al., J Biol Chem. 1996 Jul 26;271(30):17629-34. doi: 10.1074/jbc.271.30.17629.; Barleon et al., Blood. 1996 Apr 15;87(8):3336-43. PMID: 8605350.; Tchaikovski et al., Arterioscler Thromb Vasc Biol. 2008 Feb;28(2):322-8. doi: 10.1161/ATVBAHA.107.158022.; Czepluch et al., Atherosclerosis. 2011 Apr;215(2):331-8. doi: 10.1016/j.atherosclerosis.2011.01.004.), providing a mechanistic framework for the observed reduction in monocyte infiltration following SU5416 treatment.

•   The preservation of bacterial burden despite altered immune cell composition likely reflects the partial redundancy of innate immune effector mechanisms in controlling Mtb, consistent with findings from (van Crevel et al., Clin Microbiol Rev. 2002 Apr;15(2):294-309. doi: 10.1128/CMR.15.2.294-309.2002.; Court et al., J Immunol. 2010 Jun 15;184(12):7057-70. doi: 10.4049/jimmunol.1000164.; Liu et al., Cell Mol Immunol. 2017 Dec;14(12):963-975. doi: 10.1038/cmi.2017.88.; Scott et al., Infect Immun. 2002 Nov;70(11):5946-54. doi: 10.1128/IAI.70.11.5946-5954.2002.; Nouailles et al., J Clin Invest. 2014 Mar;124(3):1268-82. doi: 10.1172/JCI72030.). 

 

We have expanded the Discussion section to more explicitly address these mechanistic considerations and to clearly frame our findings within the existing literature. We agree that future studies incorporating cytokine/chemokine profiling, in vitro VEGFR blockade assays, and adoptive transfer experiments would be valuable to mechanistically validate these findings, and we have added this as a stated limitation and future direction in the revised manuscript. However, our goal is to test if the treatment limit pathology is achieved.

 

 

Major Concern 2

"Mice ranging from 8 to 28 weeks of age were used, with small experimental groups (3–6 mice per group), including both sexes. Age and sex significantly influence lung pathology, granuloma formation, and immune responses in Mtb infection. The data presented (e.g., Fig. 1) do not clarify whether animals were age- and sex-matched within experiments, and pooling data from three separate experiments introduces variability that limits interpretability."

Response to Major Concern 2

We thank the reviewer for raising this important methodological concern. We would like to clarify the following:

•   Within each individual experiment, mice were age- and sex-matched to minimize intra-experimental variability. The wide age range (8–28 weeks) reflects differences between independent experiments rather than within a single experiment.
•   We have revised the Methods section and relevant figure legends to more clearly specify the age and sex composition of animals within each individual experiment.
•   Regarding pooling of data across three independent experiments: this was performed to increase statistical power given the inherent biological variability of Mtb infection models. We have added a clearer description of the statistical approach used when combining experiments, including how inter-experimental variability was accounted for.
•   We acknowledge this as a limitation of the study and have explicitly stated this in the revised manuscript, noting that future studies with larger, strictly age- and sex-matched cohorts are warranted.

 

Major Concern 3

"The primary experiments appear to use an acute infection model, which does not fully represent chronic adaptive immune responses, despite claims regarding modulation of host immunity."

Response to Major Concern 3

We appreciate this point. We agree that the RAG1KO model, by definition, lacks functional adaptive immunity (T and B cells), making it inherently an acute/innate immunity-focused model. A  chronic  stage is not reached as the animals die. We wish to clarify that:

•   Our claims regarding host immunity modulation were intended to refer specifically to innate immune cell recruitment (monocytes, NK cells, neutrophils), not adaptive immune responses.
•   We have revised the relevant sections of the Introduction and Discussion to more carefully and precisely describe the immunological context of our findings, avoiding overstated claims about adaptive immunity modulation.
•   We further note that to complement the RAG1KO findings, we included experiments in immunocompetent C57BL/6 and B6.C3H-sst1 mice, the latter representing a chronic infection model (16-week infection), which partially addresses concerns about chronic disease representation.
•   We have updated the Discussion to more explicitly acknowledge the limitations of the acute model in representing the full spectrum of TB immunopathology.

 

Major Concern 4

"Lung pathology assessment is based on lesion area measured from H&E staining of a single lung lobe, which may not comprehensively reflect total pulmonary disease burden."

Response to Major Concern 4

We thank the reviewer for this valid methodological concern. We acknowledge that assessment of a single lung lobe provides an approximation of total lung pathology rather than a comprehensive measure of pulmonary disease burden. We would like to clarify that:

•   The lobe selected for histological analysis was consistently the same lobe across all animals, ensuring comparability within and between experimental groups. Other lobes have been used for flow cytometry and CFU analysis.
•   This approach is consistent with commonly used methodologies in murine TB lung pathology studies (Gengenbacher et al., Sci Rep. 2017 Aug 18;7(1):8853. doi: 10.1038/s41598-017-09177-2.; Lovey et al., Nat Commun. 2022 Feb 16;13(1):884. doi: 10.1038/s41467-022-28506-2.; Makafe et al., Infect Immun. 2026 Mar 10;94(3):e0057425. doi: 10.1128/iai.00574-25.; Harding et al., Cell Rep. 2019 May 14;27(7):2119-2131.e6. doi: 10.1016/j.celrep.2019.04.072.), and represents a practical balance between technical feasibility and scientific rigor.
•   We have added this as an explicit limitation in the revised manuscript and suggest that future studies incorporate whole-lung sectioning or micro-CT imaging, such as performed by Ordonez et al. (Ordonez et al., Dis Model Mech. 2016 Jul 1;9(7):779-88. doi: 10.1242/dmm.025643.), which would provide a more comprehensive assessment of pulmonary disease burden.

 

Major Concern 5

"The authors equate reduced lesion area with reduced inflammation; however, immune cell infiltration alone does not define inflammatory status. No cytokines, chemokines, or other inflammatory mediators were measured to explain the unchanged bacterial load or altered immune composition."

Response to Major Concern 5

This is an excellent and well-taken point. We agree that histological lesion area and immune cell infiltration are surrogate markers of inflammation and do not fully capture the inflammatory milieu. We acknowledge that the absence of cytokine/chemokine profiling is a limitation of the current study and addressed this under Concern 1.

•   We have revised the manuscript to more carefully use terminology such as "reduced lung pathology" and "altered immune cell infiltration" rather than broadly equating these findings with reduced inflammation.
•   We have added a dedicated paragraph in the Discussion acknowledging that future studies incorporating multiplex cytokine profiling (e.g., IL-6, TNF-α, IL-1β, CXCL chemokines) from lung homogenates would be essential to:

o   Characterize the inflammatory mediator landscape following VEGFR blockade

o   Explain the mechanism behind preserved bacterial control despite altered immune composition

•   We agree that this represents a significant area for follow-up investigation and have framed it as such.
•   At the same time it is well accepted that large lesion size correlates with more inflammation and organ damage rather than with protection (Flynn et al., Mucosal Immunol. 2011 May;4(3):271-8. doi: 10.1038/mi.2011.14.; Mehra et al., J Infect Dis. 2013 Apr;207(7):1115-27. doi: 10.1093/infdis/jis778.; Amaral et al., 2021 Feb 20;34(6):471-485. doi: 10.1089/ars.2020.8124.).

Major Concern 6

"Figures 3G and 4 include small sample sizes (n=3–4 mice), but the sex distribution of these animals is not specified."

Response to Major Concern 6

We thank the reviewer for this observation. We have updated the figure legends for Figures 3G and 4 to explicitly state the sex distribution of the animals included in these experiments. We acknowledge that the small sample sizes in these figures are a limitation and have clearly stated this in the Limitations section of the revised manuscript.

 

Major Concern 7

"In the chronic infection model (15-week-old B6.C3H-sst1 mice, N=4/group, both sexes, infected for 16 weeks), SU5416 modestly increased neutrophils (p=0.05) without affecting lesion size. Given known sex-based differences in granuloma formation, these results require stratified analysis and discussion."

Response to Major Concern 7

We appreciate this important methodological suggestion. Regarding sex-stratified analysis:

•   Given the small sample size (N=4/group), a formal sex-stratified statistical analysis is underpowered and would not yield reliable conclusions. We have explicitly acknowledged this limitation in the revised manuscript.
•   However, we have added a supplementary table presenting the individual data points with sex annotations for transparency, allowing readers to visually assess potential sex-related trends.
•   We have expanded the Discussion to address known sex-based differences in granuloma formation and immune responses in TB (Hertz et al., Semin Immunopathol. 2019 Mar;41(2):225-237. doi: 10.1007/s00281-018-0725-6. ; Dibbern et al., Sci Rep. 2017 Sep 8;7(1):10957. doi: 10.1038/s41598-017-11438-z.) and to contextualize our findings within this framework.
•   We agree that future studies with adequately powered, sex-stratified cohorts in the B6.C3H-sst1 model are needed, and we have included this as a specific recommendation for future work.

Major Concern 8

"The manuscript does not adequately address limitations, including small sample sizes, mixed age groups, sex effects, and lack of mechanistic insight."

Response to Major Concern 8

We fully accept this criticism. We have substantially expanded the Limitations section of the revised manuscript to explicitly and transparently address:

1. Small sample sizes and their impact on statistical power and generalizability
2. Mixed age groups across experiments and the potential influence of age on immune responses and lung pathology in Mtb infection
3. Sex effects and the need for future sex-stratified analyses
4. Lack of mechanistic experiments and the descriptive/exploratory nature of the current study
5. Single lobe histological assessment as a surrogate for total lung pathology
6. Absence of inflammatory mediator profiling

We believe these additions significantly improve the transparency and scientific integrity of the manuscript. We hope that these revisions adequately address Reviewer 1's concerns and that the manuscript is now considered suitable for publication. We remain available to provide any further clarification.

Reviewer 2 Report

Comments and Suggestions for Authors

In this article authors stated that MTB poses a major public health challenge, causing many deaths worldwide, particularly affecting immunocompromised individuals. As evidence, they provided previous research studies that indicate blocking VEGFR1 limits Mtb-induced pathology in immunocompetent mice. In this study they assess the VEGFR1/2 blockade in immunocompromised RAG1KO mice, showing that treatment with the VEGFR1/2 blocker SU5416 (semaxanib) reduced monocyte infiltration in the lungs without compromising bacterial protection, while improving lung pathology. Additionally, fewer NK cells were found, and an increase in neutrophil granulocytes occurred without worsening lung issues, as most remained in lung vasculature. These findings support further exploration of VEGFR blockers like SU5416 as adjunct therapies in treating tuberculosis among immunocompromised patients.

Here are some of the comments:

1. Did you check the expression (protein or mRNA) of RAGE receptors, as they have important role in inflammation?
2. There is reduced NK cell populations but functional validation such as cytokine production especially, TNF-a, IFN-g, IL-4 and IL-10 would strengthen interpretation.
3. Provide details in discussion of short treatment window of the study, is there any antibiotic co-treatment done, please justify.
4. Did the symptoms of extra-pulmonary examined during the study? Cultures of the tissues may be performed to evaluate migration.

Author Response

Response to Reviewer 2

 

We sincerely thank Reviewer 2 for their positive and constructive evaluation of our manuscript, and for providing a clear and fair summary of our study's key findings. We are pleased that the reviewer found the introduction, methods, and conclusions to be of adequate quality. We address each of their specific comments below.

 

Response to Specific Comments

 

Comment 1: "Did you check the expression (protein or mRNA) of RAGE receptors, as they have an important role in inflammation?"

 

Response to Comment 1:

We thank the reviewer for raising this interesting and relevant point. RAGE (Receptor for Advanced Glycation End-products) is indeed an important pattern recognition receptor involved in inflammatory signaling, and its role in tuberculosis-associated lung pathology is an emerging area of interest.

 

However, in the current study:

- RAGE receptor expression (protein or mRNA) was not assessed, as the primary focus of the study was on the role of VEGFR1/2 signaling in modulating immune cell recruitment and lung pathology in Mtb-infected immunocompromised mice.

- We acknowledge that RAGE signaling is one of the many factors that may contribute to the inflammatory environment observed in Mtb-infected lungs, and its potential interaction with VEGFR-mediated pathways represents an intriguing area for future investigation.

 

We have added a paragraph in the Discussion acknowledging the potential relevance of RAGE receptor signaling in TB-associated lung inflammation and proposing it as a future research direction, including both:

- Protein-level assessment (e.g., immunohistochemistry, ELISA)

- mRNA expression profiling (e.g., qRT-PCR) in lung tissue from SU5416-treated and control Mtb-infected mice

 

We thank the reviewer for this valuable suggestion, which broadens the scientific context of our findings.

 

 

Comment 2

"There is reduced NK cell populations but functional validation such as cytokine production especially, TNF-α, IFN-γ, IL-4 and IL-10 would strengthen interpretation."

 

Response to Comment 2

We fully agree with the reviewer that functional validation of the observed NK cell reduction would significantly strengthen the interpretation of our findings. In the current study:

 

- NK cell assessment was limited to quantification of cell numbers by flow cytometry, without functional characterization of cytokine production.

- We acknowledge that understanding whether the reduction in NK cell numbers following SU5416 treatment is accompanied by altered cytokine production profiles, particularly: TNF-α (pro-inflammatory, antimycobacterial), IFN-γ (critical for macrophage activation and Mtb control), IL-4 (anti-inflammatory, Th2-associated), IL-10 (immunoregulatory, anti-inflammatory) would be essential to determine whether the functional NK cell response is preserved, enhanced, or diminished following VEGFR blockade. However, the current manuscript is the first preclinical study of its kind to support further translational research relevant for immunocompromised patients. 

In response to this important point, we have expanded the Discussion to 

1. Acknowledge this as an important limitation of the current study and 
2. Discuss the potential functional implications of reduced NK cell numbers in the context of Mtb infection and VEGFR blockade.

 

 

Comment 3

"Provide details in discussion of the short treatment window of the study, is there any antibiotic co-treatment done, please justify."

 

Response to Comment 3 

We thank the reviewer for highlighting this important point regarding the treatment window and potential antibiotic co-treatment.

 

Regarding the treatment window:

We acknowledge that the SU5416 treatment window used in this study represents a relatively short intervention period in the context of TB disease progression. This was a deliberate design choice to assess the acute effects of VEGFR1/2 blockade on innate immune cell recruitment and early lung pathology and minimize potential confounding effects of prolonged VEGFR blockade on vascular homeostasis and wound healing.

We have expanded the Discussion to explicitly address this limitation, acknowledging that:

  - A short treatment window may not capture the long-term effects of VEGFR blockade on lung pathology, fibrosis, or granuloma stability.

  - Future studies employing extended treatment regimens at different stages of infection in immunocompetent animals (early vs. established disease) would be valuable to determine the optimal therapeutic window.

 

Regarding antibiotic co-treatment:

This is an important point. No antibiotic co-treatment was administered during this study. The study was designed as a proof-of-concept investigation of VEGFR blockade as a host-directed therapy (HDT), independent of antibiotic treatment, to isolate the immunomodulatory effects of SU5416 from direct antibacterial effects. We acknowledge that in a clinical context, VEGFR blockers would be used as adjunctive therapy alongside standard anti-tuberculosis antibiotics (e.g., isoniazid, rifampicin). The interaction between SU5416 and antibiotic therapy on immune responses and lung pathology remains to be investigated. We have added this justification to the Discussion and proposed combination studies (SU5416 + standard anti-TB regimen) as an important next step toward clinical translation.

 

 

Comment 4

"Did the symptoms of extra-pulmonary examined during the study? Cultures of the tissues may be performed to evaluate migration."

 

Response to Comment 4

We thank the reviewer for raising this important point regarding extra-pulmonary dissemination of Mtb. This is particularly relevant in the context of immunocompromised RAG1KO mice, which lack functional T and B cells and may be more susceptible to Mtb dissemination beyond the lungs.

 

In the current study:

- The primary focus was on pulmonary disease, and systematic evaluation of extra-pulmonary organs (e.g., spleen, liver, kidneys, lymph nodes) for Mtb dissemination was not performed.

We acknowledge this as a significant limitation, particularly given that:

  - RAG1KO mice lack adaptive immunity, potentially facilitating hematogenous spread of Mtb

  - VEGFR blockade with SU5416 could theoretically influence vascular permeability and thereby alter the pattern of Mtb dissemination

 

We have updated the Discussion and Limitations sections to:

1. Explicitly acknowledge the absence of extra-pulmonary assessment as a limitation
2. Recommend that future studies include:

   - Bacterial culture of spleen, liver, and lymph nodes to quantify extra-pulmonary bacterial burden

   - Histological examination of extra-pulmonary organs for evidence of granuloma formation or tissue damage

   - Assessment of whether SU5416 treatment influences the pattern or extent of Mtb dissemination in immunocompromised hosts.

 We agree that these analyses would provide a more complete picture of the safety and efficacy of VEGFR blockade in the context of systemic Mtb infection.

 

We are grateful to Reviewer 2 for their thoughtful and constructive feedback, which has meaningfully improved the scientific completeness and transparency of our manuscript. We hope that these revisions satisfactorily address all raised concerns.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed all the concerns and acknowledged the limitations in the discussion section. The manuscript can be accepted.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have provided a systematic and well-reasoned response to comments. The clarifications and additions to the “Discussion” section appropriately address the concerns, particularly key limitations (e.g., lack of RAGE expression analysis, absence of NK cell functional validation, short treatment window, and lack of extrapulmonary evaluation) while outlining relevant future directions. The justifications for study design selections are reasonable, and the revisions in this version improve the overall clarity and scientific context of the study in this manuscript. Based on the responses and revisions provided, the manuscript is suitable to proceed to the next stage of the publication process.