IL-13 deficiency exacerbates lung damage and impairs epithelial-derived type 2 molecules during nematode infection

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   Dec 14, 2020

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   Reply to the reviewers

   Reviewer #1 (Evidence, reproducibility and clarity (Required)):*

   This study addresses the role IL-13 in promoting lung damage following migration of the helminth N. brasiliensis larvae from the circulatory system to the lung. The work clearly shows using IL13-/- mice that Nb elicited IL-13 immunity at days 2-6 post-infection reduces pathology. The authors demonstrate an association with reduced eosinophils but no effect on neutrophil numbers.

   Proteomic analysis identifies a number of molecules known to be involved in protecting against type 2 pathologies such as relm-a and SP-D.

   The authors then identify a clear requirement for IL-13 in driving relm-a …

   Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

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   Reply to the reviewers

   Reviewer #1 (Evidence, reproducibility and clarity (Required)):*

   This study addresses the role IL-13 in promoting lung damage following migration of the helminth N. brasiliensis larvae from the circulatory system to the lung. The work clearly shows using IL13-/- mice that Nb elicited IL-13 immunity at days 2-6 post-infection reduces pathology. The authors demonstrate an association with reduced eosinophils but no effect on neutrophil numbers.

   Proteomic analysis identifies a number of molecules known to be involved in protecting against type 2 pathologies such as relm-a and SP-D.

   The authors then identify a clear requirement for IL-13 in driving relm-a expression.

   Finally, the authors present a whole lung RNA transcript profile which largely supports their proteomic observations.

   Taken together the work presents a sound case for IL-13 being an important player in protecting against initial lung pathology.

   **Major requests:**

   The paper is really very interesting and important. To an extent it questions existing dogma of IL-13 being a driver of lung inflammation.

   Addressing the following could hopefully be achieved using archived samples or with an acceptable amount of extra experimental work.

   Figure 1: D2 and D6 Lung IL-13 concentrations (ELISA) in WT mice would set the scene for the papers story*

   We agree that showing IL-13 concentrations in the lung would nicely set the stage for the role of IL-13 during ____Nippostrongylus____ infection. In the current paper, we showed IL-13 mRNA levels in Figure 3 but in a revised version, we will include D2 and D6 mRNA data in Figure 1. We attempted to quantify IL-13 protein levels in the BAL fluid of infected WT mice on D2 and D6 post-infection. However, IL-13 in the BAL was below the levels of detection for our ELISA assay. Therefore, we would need to measure IL-13 protein in total lung homogenates but we do not have material archived at present. If the editor feels this is a critical piece of data we will perform repeat experiments.

   *Figure 2: The authors should add evidence that function/activity of neutrophils/eosinophils is changed/not changed: e.g. granzyme, MBP, EPO release in BAL and/or lung. *

   __As supported by referee 3, we feel that measuring functional readouts of neutrophils and eosinophils, while interesting, is currently outside of the scope of the paper. Further, with respect to eosinophils, we see a major reduction in total eosinophil numbers in IL-13-deficient mice which would likely result in a reduction in the level of functional molecules such as MBP. Thus, these readouts in the BAL may not be a reliable indicator of cellular function and results difficult to interpret in light of altered cell numbers. __

   Additionally, some data showing changes in epithelial stress related cytokines such as IL-23 and IL-33 would be informative (IHC and /or ELISA).

   __The reviewer makes a good suggestion that would complement our proteomics/pathway analysis. As described in our comment below regarding Foxa2 pathways, we do have additional data showing epithelial cell defects in the absence of IL-13 and will add this to a revised manuscript. While we do see a trend for a reduction in IL-33 mRNA in infected IL-13-deficient mice, it is difficult to correlate this with functional protein. If requested, we can perform additional analyses to measure IL-23 and/or IL-33 protein levels in archived BAL fluid or by IHC of lung sections. __

   *The following will require a new experiment:

   The authors present a strong case for RELMa being associated with/driven by IL-13 responses. The following I feel would prove that IL-13 driven RELMa is important in reducing lung pathology. Can enhanced lung pathology or cell responses associated with pathology be reduced/altered by dosing Nb infected IL13-/- mice with recombinant relma or by restimulating BAL cells (for example) from IL-13-/- mice. This team is well placed to comment on the potential for such an in vivo experiment to be feasible.

   Or could the authors could also test the ability for other candidate molecules to reduce lung pathology? Would for example i/n dosing of IL-13-/- mice with AMCase, BRP39 or SP-D protect against pathology? It would be expected to be the case for SP-D.*

   Our previous study has shown that RELM-____a____ plays an important yet highly complex role during lung repair (see Sutherland et al. 2018: ____https://doi.org/10.1371/journal.ppat.1007423____). The suggested experiments would advance our understanding of the function of RELM-____a____ and other effector molecules during type 2 immunity and repair. However, it is unlikely that the impact of IL-13 will be due to a single effector molecule (as supported by Reviewer 3) and thus these types of experiments would shift the focus of the paper from the impact of IL-13 to understanding specific function of type 2 effectors. Since our study deals more broadly with the function of IL-13 rather than the downstream effectors, we hope that this will open up further investigation of these specific molecules to the wider community to take forward.

   *Reviewer #1 (Significance (Required)):

   The manuscript places IL-13 as an important initiator of early protection from acute lung damage. This is important as it is to an extent a non-canonical role for this cytokine. This is also important as IL-13 can be manipulated therapeutically. To maximise potential application of such drugs requires detailed understanding of the various contextual roles of IL-13. This study provides such evidence.

   The authors identify a range of target mediators.

   This is an important body of work that is useful for understanding how acute lung damage can be regulated.

   This work will be of interest to Type 2 immunologists, any researcher with an interest in pulmonary inflammation as well as mucosal immunity.

   I make these suggestions/comments based on my own background in Type 2 immunity, lung inflammation and parasitic helminth infection and immunity.

   Reviewer #2 (Evidence, reproducibility and clarity (Required)):

   In this manuscript, Chenery et al report that IL-13 plays a critical role in protecting mice from lung damage caused by the infection of a nematode, Nippostrongylus Brasiliensis, in WT or IL-13 knockout mice (IL-13 eGFP knock-in mice, Neill et al., Nature 2010). Phenotypically, they demonstrated that IL-13 genetic deficiency resulted in more severe lung injuries and haemorrhaging following the larvae migratory infections. Through the proteomic and transcriptomic profiling, they identified gene-expression changes involved in the cellular stress responses, e.g. up-regulating the expression of epithelial-derived type 2 molecules, controlled by IL-13. They also found that type 2 effector molecules including RELM-alpha and surfactant protein D were compromised in IL-13 knockout mice. Thus, they proposed that IL-13 has tissue-protective functions during lung injury and regulates epithelial cell responses during type 2 immunity in this acute setting. Overall, the manuscript was clearly written and a number of findings were interesting and expected compared to the published knowledge. However, this work could be improved and more impactful by further performing the following suggested experiments.

   Major points:

   1. It may not be accurate to claim that "IL-13 played a critical role in limiting tissue injury ... in the lung following infection" since IL-13 participates in both repelling worms and activating tissue reparative responses. It is very hard to distinguish these two kinds of responses with the current experimental settings because the much higher worm burden led to more direct lung damage in IL-13-/- mice than WT counterparts.*

   The reviewer raises an important point that we will need to clarify in a revised manuscript. Based on several studies, the role of IL-13 in mediating ____Nippostrongylus____ expulsion occurs in the small intestine, after the parasites have already cleared the lung tissue. The number of worms in the lung do not differ at the time points we are investigating. We have qRT-PCR data measuring ____Nippostrongylus____-specific actin levels, which we and others have previously shown to accurately reflect worm numbers. We can therefore demonstrate that the differences in lung damage do not reflect a difference in the number of larvae in the lungs of IL-13 KO mice compared to WT mice. These data will be incorporated into the manuscript to better clarify this point.

   1. It would be more informative if the authors could perform the RNA-seq analysis on the IL-13-responsive cell type such as airway epithelial cells (goblet cell) by comparing WT vs IL13-/- in the context of lung damage caused by Nitrostrongylus Brasiliensis infection.*

   __RNA-sequencing of specific cells would indeed be an excellent experiment that would reveal more IL-13-depedendent processes in our model. However, this would be a considerable undertaking at this stage (as reviewer 3 has pointed out). Nonetheless, our extended analysis of the Foxa2 pathway as requested below has highlighted a number of genes regulated by IL-13, which are known to be involved in epithelial cell function. __

   3. Figure 6C, the transcriptional profiling of mouse lungs revealed that the Foxa2 pathway was significantly up-regulated in the IL-13-/- infected mice. This is an important finding because this pathway plays a critical role in the process of alveolarization and inhibiting goblet cell hyperplasia. In order to validate this finding, some components in this pathway could be further examined.

   We agree with the reviewer that showing additional validation data to support the Foxa2 defect in IL-13-deficient mice would strengthen our paper’s overall message. We have additional qRT-PCR data of IL-13-dependent genes regulated by Foxa2 (____Clca1____, ____Muc5ac____, ____Ccl11____, and ____Foxa3____) that clearly support this epithelial cell-specific defect that we can readily incorporate into the revised paper.

   *Reviewer #2 (Significance (Required)):

   Overall, the manuscript was clearly written and a number of findings were interesting and expected compared to the published knowledge.

   **Referees cross-commenting**

   To Reviewer #1's Review: fair and constructive

   To Reviewer #3's Review: agree in general.*

   Reviewer #3 (Evidence, reproducibility and clarity (Required)):

   In this study, Allen, Sutherland and colleagues utilize IL-13 deficient mice to investigate the function of IL-13 in the early response to lung tissue damage induced by helminth infection. They demonstrate that IL-13 deficiency has significant effects on the acute tissue response to helminth infection (at day 2 and 6 post-infection). Particularly, IL-13 deficiency results in increased lung hemorrhaging, and more pronounced lung tissue damage evidenced by increased gaps in the alveolar architecture. They perform proteomic and transcriptomic profiling of the lungs to determine IL-13-induced pathways and demonstrate many protein and gene expression changes in the absence of IL-13. These include dysregulated collagens, reduced epithelial-derived proteins RELMalpha and surfactant protein D, downregulated pathways related to cellular stress, and increased genes associated with the Foxa2 pathway.

   Overall, the key conclusions are convincing, and the study design, methods and data analysis are clear, rigorous and thorough.

   **Minor Comments:**

   1. The authors concluded that lung epithelial cells are more sensitive to IL-13 than IL-4, but the intranasal injection of both proteins showed a similar induction of RELMα - investigation into this difference would be useful. Alternatively, providing an explanation for these different findings could be helpful.*

   __Our suggestion that epithelial cells are likely to be more sensitive to IL-13 was based both on our data and the existing literature. We would agree that we do not have definitive evidence for this. Indeed, because the type 2 receptor can respond to both IL-4 and IL-13 this issue is difficult to easily resolve experimentally. We will expand on this in a revised manuscript, making our explanations clearer whilst acknowledging the alternative explanations. __

   2. Providing data by immunofluorescence or flow cytometry of non-epithelial expression of RELMalpha following intranasal versus intraperitoneal injection of IL-4 versus IL-13 would be useful.

   This is a good suggestion and we have additional flow cytometry data looking at hematopoietic cell expression of RELM-____a____ from these experiments that we can incorporate into the revised manuscript. We have found that airway macrophages were another source of RELM-____a____ in the lung and mirrored the airway epithelial cell responses to both intraperitoneal and intranasal delivery of IL-4 and IL-13.

   3. Discussion of IL-13Ra1 deficient mice would be useful, in particular the study by Karo-Atar and Munitz in Mucosal Immunology 2016, showing that IL13Ra1 is protective against bleomycin-induced pulmonary injury (PMID: 26153764). Comparing their data with the gene expression datasets from this study would be useful (acknowledging the caveat that IL-4 effects through the type 2 receptor would also be abrogated in these IL13Ra1 mice).

   We agree that a comparison of IL-13Ra1 versus IL-13 deficiency should be included in the discussion of our manuscript. These authors found epithelial-specific defects in IL-13Ra1-deficient mice such as Clca1 (aka Clca3), RELM-____a____, and chitinase-like proteins even under homeostatic conditions, which is highly consistent with our data. This study also found that IL-13Ra1 deficiency led to increased bleomycin-induced pathology and together with our data, offers further insight into the IL-13/IL-13R____a____1 axis during lung injury. We will add these points to our discussion and will attempt to directly compare their gene expression data set with our data to find more overlapping genes between the two mouse strains and disease models.

   4. The authors reference Chen et al. Nature Medicine 2012, but do not discuss the finding in this paper that neither IL-4-/- nor IL13Ra1-/- have increased lung hemorrhage. This might be a mouse strain issue and worthwhile discussing.

   __This is indeed a very important point we will address in a revised discussion. IL-4R____a____-deficient mice did show increased bleeding in the Chen et al. study that was not seen in the IL-13R____a____1 KO suggesting IL-4 alone is sufficient to limit bleeding. This is in contrast to our study where we found increased bleeding in IL-13 KO mice independent of IL-4. However, a major difference between the studies is the background strain of mice used, which was BALB/c in the Chen et al. study versus C57BL/6 mice we used in our study. In addition to differences in IL-4 and IL-13 levels between the strains, we have unpublished observations of major differences in vascular integrity with BALB/c much more prone to bleeding, which is an active area of investigation in the lab. Although we have yet to unravel these differences mechanistically, they could explain differential requirements for IL-4 versus IL-13 to limit bleeding between the two strains. __

   5. Reference 32 and 36 (Sutherland PLoS pathogens) are duplicates

   Our apologies, we will fix the reference duplication.

   *Reviewer #3 (Significance (Required)):

   This study addresses the specific function of IL-13 in acute helminth infection of the lung, which has not previously been studied, as most studies investigate the combined function of IL-4 and IL-13 through IL-4 receptor KO or Stat6 KO mice.

   It is a thorough, well-conducted and well-organized study with high quality data using 'omics' strategies to profile IL-13-induced genes and proteins. Their data identifies intriguing pathways that are dependent on IL-13, opening new avenues to explore for IL-13-mediated protective roles in acute lung tissue damage. Therefore this study provides conceptual and technical advances. Additionally, since targeting IL-4 and IL-13 are in clinical trials or employed therapeutically for pulmonary disorders, the findings from these studies are clinically relevant. It would however have been useful to validate some of these pathways and demonstrate epithelial-specific outcomes for IL-13-induced tissue protection.

   Previous studies using IL4RKO have shown that IL-4 and IL-13 are necessary to protect from acute tissue damage in helminth infection (Chen, Nature medicine - referred to by authors). Other studies have investigated IL-13 in fibrosis and granulomatous inflammation (papers referenced by authors, and Ramalingam Nature Immunology 2009). Last, one study shows that IL-13Ra1 signaling is important for protection in bleomycin-induced lung injury, findings using a different transgenic mouse, which are relevant for this study and may be useful to discuss (Karo-Atar, Mucosal Immunology 2016).

   As stated above - the data in this manuscript identify intriguing pathways that are dependent on IL-13, opening new, exciting avenues to explore for IL-13-mediated protective roles in acute lung tissue damage. Their data is also unique as it combines proteomics and transcriptomics, and identities previously unappreciated IL-13 regulated pathways such as cellular stress and Foxa2, which would be interesting to investigate further.

   **Referees cross-commenting**

   To Reviewer 1: The suggested data for Figure 1 (IL-13 concentrations) could be useful, but suggested experiments for Figure 2 could be outside the main focus of the paper.

   For the main suggested experiment: treatment of IL-13-/- with RELMalpha, this could be useful, One caveat is that RELMalpha might not be the only effector molecule downstream of IL-13 so the authors may not get a definitive answer. An alternative (although not as RELMalpha-specific) would be to treat IL13KO mice with FcIL-4 or FcIL-13 - the latter that drives RELMalpha, and look at whether FcIL-13 is more protective than FcIL-4.*

   We agree that rescue experiments could provide insights into the relative protective effects of IL-4 versus IL-13. However, it might be challenging to interpret the results in part because of the difficulty in establishing physiologically relevant doses and timing and the fact that IL-4 will also signal through the type 2 receptor. These difficulties are reflected in the interpretation of our current data as discussed above (pt. 1 reviewer 3). Although we have found IL-4 and IL-13 delivery experiments valuable and have used them in many of our papers, we have always been cautious in our interpretation, as we typically use these at super-physiological doses. However, this is an experiment we would consider if the editors felt it essential to the story.

   To Reviewer 2: I agree with points 1 and 3 - especially with point 3, which would give more in-depth understanding into the functional outcomes of the IL-13 -> FoxA2 pathway identified. For point 2, RNA-seq of epithelial cells would be informative, but may be beyond the scope of the project.

   Read the original source
2. 

   Review Commons

   Dec 14, 2020

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #3

   Evidence, reproducibility and clarity

   In this study, Allen, Sutherland and colleagues utilize IL-13 deficient mice to investigate the function of IL-13 in the early response to lung tissue damage induced by helminth infection. They demonstrate that IL-13 deficiency has significant effects on the acute tissue response to helminth infection (at day 2 and 6 post-infection). Particularly, IL-13 deficiency results in increased lung hemorrhaging, and more pronounced lung tissue damage evidenced by increased gaps in the alveolar architecture. They perform proteomic and transcriptomic profiling of the lungs to determine IL-13-induced pathways and demonstrate many protein and …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #3

   Evidence, reproducibility and clarity

   In this study, Allen, Sutherland and colleagues utilize IL-13 deficient mice to investigate the function of IL-13 in the early response to lung tissue damage induced by helminth infection. They demonstrate that IL-13 deficiency has significant effects on the acute tissue response to helminth infection (at day 2 and 6 post-infection). Particularly, IL-13 deficiency results in increased lung hemorrhaging, and more pronounced lung tissue damage evidenced by increased gaps in the alveolar architecture. They perform proteomic and transcriptomic profiling of the lungs to determine IL-13-induced pathways and demonstrate many protein and gene expression changes in the absence of IL-13. These include dysregulated collagens, reduced epithelial-derived proteins RELMalpha and surfactant protein D, downregulated pathways related to cellular stress, and increased genes associated with the Foxa2 pathway.

   Overall, the key conclusions are convincing, and the study design, methods and data analysis are clear, rigorous and thorough.

   Minor Comments:

   1. The authors concluded that lung epithelial cells are more sensitive to IL-13 than IL-4, but the intranasal injection of both proteins showed a similar induction of RELMα - investigation into this difference would be useful. Alternatively, providing an explanation for these different findings could be helpful.
   2. Providing data by immunofluorescence or flow cytometry of non-epithelial expression of RELMalpha following intranasal versus intraperitoneal injection of IL-4 versus IL-13 would be useful.
   3. Discussion of IL-13Ra1 deficient mice would be useful, in particular the study by Karo-Atar and Munitz in Mucosal Immunology 2016, showing that IL13Ra1 is protective against bleomycin-induced pulmonary injury (PMID: 26153764). Comparing their data with the gene expression datasets from this study would be useful (acknowledging the caveat that IL-4 effects through the type 2 receptor would also be abrogated in these IL13Ra1 mice).
   4. The authors reference Chen et al. Nature Medicine 2012, but do not discuss the finding in this paper that neither IL-4-/- nor IL13Ra1-/- have increased lung hemorrhage. This might be a mouse strain issue and worthwhile discussing.
   5. Reference 32 and 36 (Sutherland PLoS pathogens) are duplicates

   Significance

   This study addresses the specific function of IL-13 in acute helminth infection of the lung, which has not previously been studied, as most studies investigate the combined function of IL-4 and IL-13 through IL-4 receptor KO or Stat6 KO mice.

   It is a thorough, well-conducted and well-organized study with high quality data using 'omics' strategies to profile IL-13-induced genes and proteins. Their data identifies intriguing pathways that are dependent on IL-13, opening new avenues to explore for IL-13-mediated protective roles in acute lung tissue damage. Therefore this study provides conceptual and technical advances. Additionally, since targeting IL-4 and IL-13 are in clinical trials or employed therapeutically for pulmonary disorders, the findings from these studies are clinically relevant. It would however have been useful to validate some of these pathways and demonstrate epithelial-specific outcomes for IL-13-induced tissue protection.

   Previous studies using IL4RKO have shown that IL-4 and IL-13 are necessary to protect from acute tissue damage in helminth infection (Chen, Nature medicine - referred to by authors). Other studies have investigated IL-13 in fibrosis and granulomatous inflammation (papers referenced by authors, and Ramalingam Nature Immunology 2009). Last, one study shows that IL-13Ra1 signaling is important for protection in bleomycin-induced lung injury, findings using a different transgenic mouse, which are relevant for this study and may be useful to discuss (Karo-Atar, Mucosal Immunology 2016).

   As stated above - the data in this manuscript identify intriguing pathways that are dependent on IL-13, opening new, exciting avenues to explore for IL-13-mediated protective roles in acute lung tissue damage. Their data is also unique as it combines proteomics and transcriptomics, and identities previously unappreciated IL-13 regulated pathways such as cellular stress and Foxa2, which would be interesting to investigate further.

   Referees cross-commenting

   To Reviewer 1: The suggested data for Figure 1 (IL-13 concentrations) could be useful, but suggested experiments for Figure 2 could be outside the main focus of the paper.

   For the main suggested experiment: treatment of IL-13-/- with RELMalpha, this could be useful, One caveat is that RELMalpha might not be the only effector molecule downstream of IL-13 so the authors may not get a definitive answer. An alternative (although not as RELMalpha-specific) would be to treat IL13KO mice with FcIL-4 or FcIL-13 - the latter that drives RELMalpha, and look at whether FcIL-13 is more protective than FcIL-4.

   To Reviewer 2: I agree with points 1 and 3 - especially with point 3, which would give more in-depth understanding into the functional outcomes of the IL-13 -> FoxA2 pathway identified. For point 2, RNA-seq of epithelial cells would be informative, but may be beyond the scope of the project.

   Read the original source
3. 

   Review Commons

   Dec 14, 2020

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   In this manuscript, Chenery et al report that IL-13 plays a critical role in protecting mice from lung damage caused by the infection of a nematode, Nippostrongylus Brasiliensis, in WT or IL-13 knockout mice (IL-13 eGFP knock-in mice, Neill et al., Nature 2010). Phenotypically, they demonstrated that IL-13 genetic deficiency resulted in more severe lung injuries and haemorrhaging following the larvae migratory infections. Through the proteomic and transcriptomic profiling, they identified gene-expression changes involved in the cellular stress responses, e.g. up-regulating the expression of epithelial-derived type 2 molecules, …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   In this manuscript, Chenery et al report that IL-13 plays a critical role in protecting mice from lung damage caused by the infection of a nematode, Nippostrongylus Brasiliensis, in WT or IL-13 knockout mice (IL-13 eGFP knock-in mice, Neill et al., Nature 2010). Phenotypically, they demonstrated that IL-13 genetic deficiency resulted in more severe lung injuries and haemorrhaging following the larvae migratory infections. Through the proteomic and transcriptomic profiling, they identified gene-expression changes involved in the cellular stress responses, e.g. up-regulating the expression of epithelial-derived type 2 molecules, controlled by IL-13. They also found that type 2 effector molecules including RELM-alpha and surfactant protein D were compromised in IL-13 knockout mice. Thus, they proposed that IL-13 has tissue-protective functions during lung injury and regulates epithelial cell responses during type 2 immunity in this acute setting. Overall, the manuscript was clearly written and a number of findings were interesting and expected compared to the published knowledge. However, this work could be improved and more impactful by further performing the following suggested experiments.

   Major points:

   1. It may not be accurate to claim that "IL-13 played a critical role in limiting tissue injury ... in the lung following infection" since IL-13 participates in both repelling worms and activating tissue reparative responses. It is very hard to distinguish these two kinds of responses with the current experimental settings because the much higher worm burden led to more direct lung damage in IL-13-/- mice than WT counterparts.
   2. It would be more informative if the authors could perform the RNA-seq analysis on the IL-13-responsive cell type such as airway epithelial cells (goblet cell) by comparing WT vs IL13-/- in the context of lung damage caused by Nitrostrongylus Brasiliensis infection.
   3. Figure 6C, the transcriptional profiling of mouse lungs revealed that the Foxa2 pathway was significantly up-regulated in the IL-13-/- infected mice. This is an important finding because this pathway plays a critical role in the process of alveolarization and inhibiting goblet cell hyperplasia. In order to validate this finding, some components in this pathway could be further examined.

   Significance

   Overall, the manuscript was clearly written and a number of findings were interesting and expected compared to the published knowledge.

   Referees cross-commenting

   To Reviewer #1's Review: fair and constructive

   To Reviewer #3's Review: agree in general.

   Read the original source
4. 

   Review Commons

   Dec 14, 2020

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   This study addresses the role IL-13 in promoting lung damage following migration of the helminth N. brasiliensis larvae from the circulatory system to the lung. The work clearly shows using IL13-/- mice that Nb elicited IL-13 immunity at days 2-6 post-infection reduces pathology. The authors demonstrate an association with reduced eosinophils but no effect on neutrophil numbers.

   Proteomic analysis identifies a number of molecules known to be involved in protecting against type 2 pathologies such as relm-a and SP-D.

   The authors then identify a clear requirement for IL-13 in driving relm-a expression.

   Finally, the authors present a …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   This study addresses the role IL-13 in promoting lung damage following migration of the helminth N. brasiliensis larvae from the circulatory system to the lung. The work clearly shows using IL13-/- mice that Nb elicited IL-13 immunity at days 2-6 post-infection reduces pathology. The authors demonstrate an association with reduced eosinophils but no effect on neutrophil numbers.

   Proteomic analysis identifies a number of molecules known to be involved in protecting against type 2 pathologies such as relm-a and SP-D.

   The authors then identify a clear requirement for IL-13 in driving relm-a expression.

   Finally, the authors present a whole lung RNA transcript profile which largely supports their proteomic observations.

   Taken together the work presents a sound case for IL-13 being an important player in protecting against initial lung pathology.

   Major requests:

   The paper is really very interesting and important. To an extent it questions existing dogma of IL-13 being a driver of lung inflammation.

   Addressing the following could hopefully be achieved using archived samples or with an acceptable amount of extra experimental work.

   Figure 1: D2 and D6 Lung IL-13 concentrations (ELISA) in WT mice would set the scene for the papers story

   Figure 2: The authors should add evidence that function/activity of neutrophils/eosinophils is changed/not changed: e.g. granzyme, MBP, EPO release in BAL and/or lung. Additionally, some data showing changes in epithelial stress related cytokines such as IL-23 and IL-33 would be informative (IHC and /or ELISA).

   The following will require a new experiment:

   The authors present a strong case for RELMa being associated with/driven by IL-13 responses. The following I feel would prove that IL-13 driven RELMa is important in reducing lung pathology. Can enhanced lung pathology or cell responses associated with pathology be reduced/altered by dosing Nb infected IL13-/- mice with recombinant relma or by restimulating BAL cells (for example) from IL-13-/- mice. This team is well placed to comment on the potential for such an in vivo experiment to be feasible.

   Or could the authors could also test the ability for other candidate molecules to reduce lung pathology? Would for example i/n dosing of IL-13-/- mice with AMCase, BRP39 or SP-D protect against pathology? It would be expected to be the case for SP-D.

   Significance

   The manuscript places IL-13 as an important initiator of early protection from acute lung damage. This is important as it is to an extent a non-canonical role for this cytokine. This is also important as IL-13 can be manipulated therapeutically. To maximise potential application of such drugs requires detailed understanding of the various contextual roles of IL-13. This study provides such evidence.

   The authors identify a range of target mediators.

   This is an important body of work that is useful for understanding how acute lung damage can be regulated.

   This work will be of interest to Type 2 immunologists, any researcher with an interest in pulmonary inflammation as well as mucosal immunity.

   I make these suggestions/comments based on my own background in Type 2 immunity, lung inflammation and parasitic helminth infection and immunity.

   Read the original source
5. 

   Version published to 10.1101/2020.10.14.337949 on bioRxiv

   Oct 14, 2020