TRAF3 gene regulates macrophage migration and activation by lung epithelial cells infected with Aspergillus fumigatus

ABSTRACT Aspergillus fumigatus is an opportunistic pathogen that infects immunocompromised patients and imposes a heavy burden on global health. Searching for key genes in host resistance to A. fumigatus infection can help us understand the mechanisms of A. fumigatus-host interactions and provide new targets for the treatment of fungal infections. TRAF3 is one of the intracellular adapter proteins of the innate immune response that regulates signaling in various cellular processes, including host defense against pathogens. However, the defense mechanism of TRAF3 against A. fumigatus infection remains unknown. In this study, we found that TRAF3 overexpression led to the adhesion and internalization of more spores of A. fumigatus in lung epithelial cells and thus greater host immune surveillance evasion. Meanwhile, TRAF3 was able to regulate the expression of pro-inflammatory cytokines in lung epithelial cells infected with A. fumigatus through the negative regulation of NF-κB and MAPK signaling pathways. In this study, we also examined the effects of TRAF3 in the interaction between lung epithelial cells, macrophages, and A. fumigatus spores, and the results showed that TRAF3-overexpressing lung epithelial cells reduced the migration and activation of macrophages after A. fumigatus infection. In vivo experiments using TRAF3-overexpressing transgenic zebrafish larvae revealed that TRAF3 overexpression increased the fungal load and mortality of zebrafish infected with A. fumigatus. In conclusion, the TRAF3 gene can negatively regulate the resistance of lung epithelial cells to A. fumigatus, which plays an important role in the early infection processes of A. fumigatus. IMPORTANCE Aspergillus fumigatus can infect immunocompromised individuals and cause chronic and fatal invasive fungal infections. A better understanding of the molecular mechanisms of A. fumigatus-host interactions may provide new references for disease treatment. In this study, we demonstrated that the TRAF3 gene plays an important role in the early infection of A. fumigatus by regulating the resistance of lung epithelial cells to A. fumigatus. Macrophages are the most abundant innate immune cells in the alveoli; however, few studies have reported on the interactions between lung epithelial cells and macrophages in response to A. fumigatus invasion. In our study, it was demonstrated that the TRAF3 gene reduces migration to macrophages and cytokine production by negatively regulating lung epithelial cell adhesion and internalization of A. fumigatus spores. Together, our results provide new insights into lung epithelial cell-macrophage interactions during A. fumigatus infection.

fluors have nearly identical spectral profiles.
-In figures 2D and 4E, the TRAF3 OV line appears to have more fluorescence than the control in the representative images.As annexin V is a common marker for apoptosis, is it possible that viability of the OV line is altered even without spore addition, and that the down-regulation of inflammatory cytokines in later figures is simply a consequence of toxicity induced by the robust overexpression of this protein?Quantifying Annexin V fluorescence in the absence of spores and complimenting it with a viability or stress dye (like propidium iodide) would be useful in addressing this concern.
-In figure 3, the authors thoroughly showed that TRAF3 overexpression attenuated the production of key pro-inflammatory cytokines by A549 cells in response to fungal spores.Given the positive correlation between TRAF3 activation and type I interferon, it would be interesting to see if IFN-I was inversely increased in response to TRAF3 overexpression and spore stimulation.
-The authors did a nice job looking at signaling pathway activation in figure 5.However, the total levels of these proteins need to be considered as well, as the stoichiometry between activated(phosphorylated)/total protein concentration is a driving factor in these responses.Quantification (like what was done for the phosphorylated states of these proteins) showing if TRAF3 overexpression impacts total p65, IkBa, ERK and AKT levels should be included to provide justification that overexpression is impacting activation specifically, rather than simply altering the basal levels of these proteins.
-In figure 5C, quantification of nuclear translocation would help strengthen the claim that TRAF3 OV inhibits p65 nuclear translocation in response to spores.
-It would also be interesting to see what happens if fungal cells germinate following internalization.As the TRAF3 OV lines have more internalized spores, it is reasonable to assume that these cells will be more damaged during fungal germination, and consequently may increase pro-inflammatory cytokine production and macrophage recruitment later in the infection process.This information could help explain the hypervirulent phenotype observed in TRAF3 OV transgenic zebrafish as well.
Text Comments: -In line 63, the authors state "Some studies have shown that A. fumigatus infects lung epithelial cells in vitro for 6 hours".Is this specifically referring to the spores prior to germination?Clarifying this statement would be helpful.
-Figures 1A and C on the individual figure 1 PDF and in the image at the end of the compiled manuscript have an X-axis that states that spores are absent in either condition.This should be fixed to show that conditions where TRAF3 expression is reduced have both spores and A549 cells.
-In lines 368-370 in the Immunofluorescence methods section, indicate the concentration or the dilution factor for the antibodies used.
-The statistical analyses performed in each figure are difficult to assess.The authors state that one-way ANOVAs followed by ttests were used, but many figures show a comparison between only two groups (i.e.figures 1 and 2), where an ANOVA is not appropriate.The authors should more clearly state the type of statistical analysis performed throughout the paper to address this.

Reviewer #2 (Comments for the Author):
Thank you for this submission and clearly telling an interesting story of how TRAF3 regulation in response to Aspergillus fumigatus is worthy of greater appreciation and future study.
I have recommended some minor changes.Please see attached comments for details.

Staff Comments:
The submi ed manuscript by Shang et al. expands on our understanding of A. fumigatus interac ons with host lung epithelia by assessing the impact that TRAF3, a key node in immune signaling, has on spore interac ons with A549 epithelial cells in vitro.The authors nicely showed that TRAF3 expression is decreased in A549 cells in response to fungal spore exposure, which led them to hypothesize that down-regula on of this protein is important in the lung epithelial an fungal response.Accordingly, they showed that ectopic overexpression of TRAF3 increased spore adhesion and internaliza on by A549 cells and a enuated the produc on of proinflammatory cytokines in response to spore exposure.This corresponded with reduced macrophage recruitment in vitro, which they claim is the consequence of greater host immune system evasion via increased A549 internaliza on, and enhanced suscep bility to A. fumigatus infec on in transgenic zebrafish lines that overexpressed TRAF3.
Overall, the manuscript is well wri en, and the experiments are appropriately designed and controlled to answer the ques ons they sought to ask.However, I have several ques ons and sugges ons that I believe would help strengthen the claims made in this report.

General Comments
-The authors do a nice job showing that TRAF3 overexpression impacts the epithelial response to A. fumigatus spores.However, this claim would be strengthened by complimen ng overexpression data with knockdown data, or by overexpressing an inhibited TRAF3 gene as a control (possibly a gene lacking the nuclear localiza on sequence).
-How did the authors differen ate fluorescence between the eGFP signal in transfected A549 cells containing overexpression/control plasmids and the annexin V-FITC fluorescence during microscopy analyses?It is unclear and the two fluors have nearly iden cal spectral profiles.
-In figures 2D and 4E, the TRAF3 OV line appears to have more fluorescence than the control in the representa ve images.As annexin V is a common marker for apoptosis, is it possible that viability of the OV line is altered even without spore addi on, and that the down-regula on of inflammatory cytokines in later figures is simply a consequence of toxicity induced by the robust overexpression of this protein?Quan fying Annexin V fluorescence in the absence of spores and complimen ng it with a viability or stress dye (like propidium iodide) would be useful in addressing this concern.
-In figure 3, the authors thoroughly showed that TRAF3 overexpression a enuated the produc on of key pro-inflammatory cytokines by A549 cells in response to fungal spores.Given the posi ve correla on between TRAF3 ac va on and type I interferon, it would be interes ng to see if IFN-I was inversely increased in response to TRAF3 overexpression and spore s mula on.
-The authors did a nice job looking at signaling pathway ac va on in figure 5.However, the total levels of these proteins need to be considered as well, as the stoichiometry between ac vated(phosphorylated)/total protein concentra ons is a driving factor in these responses.
Quan fica on (like what was done for the phosphorylated states of these proteins) showing if TRAF3 overexpression impacts total p65, IkBa, ERK and AKT levels should be included to provide jus fica on that overexpression is impac ng ac va on specifically, rather than simply altering the basal levels of these proteins.
-In figure 5C, quan fica on of nuclear transloca on would help strengthen the claim that TRAF3 OV inhibits p65 nuclear transloca on in response to spores.
-It would be interes ng to see what happens if fungal cells germinate following internaliza on.As the TRAF3 OV lines have more internalized spores, it is reasonable to assume that these cells will be more damaged during fungal germina on, and consequently may increase proinflammatory cytokine produc on and macrophage recruitment later in the infec on process.This informa on could help explain the hypervirulent phenotype observed in transgenic zebrafish as well.
Text Comment -In line 63, the authors state "Some studies have shown that A. fumigatus infects lung epithelial cells in vitro for 6 hours".Is this specifically referring to the spores prior to germina on?Clarifying this statement would be helpful.
-Figures 1A and C on the individual figure 1 PDF and in the image at the end of the compiled manuscript have an X-axis that states that spores are absent in either condi on.This should be fixed to show that condi ons where TRAF3 expression is reduced have both spores and A549 cells.
-In lines 368-370 in the Immunofluorescence methods sec on, indicate the concentra on or the dilu on factor for the an bodies used.
-The sta s cal analyses performed in each figure are difficult to assess.The authors state that one-way ANOVAs followed by t-tests were used, but many figures show a comparison between only two groups (i.e.figure 1), where an ANOVA is not appropriate.The authors should more clearly state the type of sta s cal analysis performed throughout the paper to address this.

2023-07-26
Reviewer Summary: Here, in "TRAF3 gene regulates macrophage migration and activation by lung epithelial cells infected with Aspergillus fumigatus" the authors document a role for TRAF3 in mediating cellular (A549 and THP1) and host (zebrafish) responses to exposure and infection by the filamentous fungus Aspergillus fumigatus.Under normal conditions TRAF3 expression and protein abundance goes down in response to A. fumigatus spores, likely ensuring appropriate and functional immune response to this opportunistic pathogen.However, disturbing this normal response through upregulation of TRAF3 expression in A549 cells results in increased spore adhesion and internalization, an overall suppression of proinflammatory cytokines, and diminished interaction with alveolar macrophages.In vivo zebrafish data corroborates the in vitro data and further adds a survival component where TRAF3 overexpression results in increased host mortality.

General Comments:
Readers of this work will wonder why TRAF3 is only overexpressed and not knocked down in any of the experimental in vitro and in vivo models here.Therefore, it would be appropriate to acknowledge in the Discussion Section: 1) TRAF3 knockdown/knockout studies in the literature and how those data relate to this study, and 2) why no TRAF3 knockdown/knockout models were used here.

Figure Comments:
Figure 1 • Consider rewording the figure caption: "FIG1 TRAF3 down-regulation responds to A. fumigatus infection in lung epithelial cells".• Make non-overlay images in (1D) black/white for contrast and enhance magnification to clearly illustrate colocalization of DAPI and TRAF3 signals.
Figure 2 • Excellent figure caption title • Same image comments as for Fig. 1 -make non-overlay images black/white for contrast and only use color for overlays.Consider also changing the colors (for example, DAPI doesn't have to be dark blue) on overlay for enhanced contrast.
Figure 3 • Excellent figure caption title • Using "-" to represent "normal" TRAF3 expression is misleading as readers are more likely to interpret it as knockdown or knockout.Please use different nomenclature for TRAF3 than +/-to avoid confusion.Especially because "+/-" is used accurately to represent "presence/absence" of spores.Figure 5 • Single channel images should be black and white for contrast.Overlays can be color.
• p-ERK and p-AKT Western (5D) and quantitation (5E) data do not seem to visually agree with each other.Perhaps the blot was overexposed?Is there a better representative image?Was p-ERK and p-AKT normalized to ERK and AKT, respectively, and not GAPDH?These discrepancies between 5D and 5E undermine confident takeaways of the conclusions made from this figure.
Figure 6 • Update caption title to describe the figure's data and summary takeaway.
• Show TRAF3 relative mRNA in zebrafish uninfected with A. fumigatus spores to demonstrate overexpression in vivo.This could be supplemental data.
Figure 7 • Excellent schematic that summarizes the paper's data Supplementary Figure 1 • Caption title should summarize data and takeaway • This is a great insight.
Thank you for the reviewers' comments concerning our manuscript entitled "TRAF3 gene regulates macrophage migration and activation by lung epithelial cells infected with Aspergillus fumigatus" (ID: Spectrum02699-23).We appreciate the time and effort each of the reviewers have dedicated to providing feedback on our manuscript and are grateful for the insightful comments and valuable advice to our paper.We have studied comments carefully and we did our best to reply to the comments and we hope that it will meet your approval.Revised portion are marked in purple in the paper, for a point-by-point response to the reviewers' comments and concerns.
Responds to the reviewer's comments: Reviewer #1 General Comments -The authors do a nice job showing that TRAF3 overexpression impacts the epithelial response to A. fumigatus spores.However, this claim would be strengthened by complimenting overexpression data with knockdown data, or by overexpressing an inhibited TRAF3 gene as a control (possibly a gene lacking the nuclear localization sequence).

Response:
We agree with the comment that raised an important topic.TRAF3 knockout mice die in the early stages of life, indicating that this gene has an important biological function in postnatal development and in maintaining normal immune system function.Moreover, there are no agonists or inhibitors available to investigate the TRAF3 gene, which makes the study of the biological function of TRAF3 more difficult.First of all, in this research we found that TRAF3 gene expression was down-regulated after infection of lung epithelial cells with A. fumigatus.Considering also the difficulty of animal model construction and the consistency of in vivo and in vitro experiments.In this study, we chose to construct an in vivo and in vitro model of TRAF3 gene overexpression to in turn demonstrate the role played by TRAF3 gene in the early infection process of A. fumigatus, and added this idea to the discussion section of the article(see line 213-220).
-How did the authors differentiate fluorescence between the eGFP signal in transfected A549 cells containing overexpression/control plasmids and the annexin V-FITC fluorescence during microscopy analyses?It is unclear and the two fluors have nearly identical spectral profiles.
Response: Thank you for pointing this out.When we designed the adhesion experiment, the first choice was the spontaneous eGFP fluorescence after cell transfection with the plasmid.But after cell fixation, the eGFP fluorescence was weak, and we needed to increase the exposure time to see the fluorescence clearly, which made the cell fluorescence counting inaccurate.We wanted to find a universal fluorescent dye for labeling cell membranes.And then we associated that Annexin V can label the cell membrane of dead cells, while cells die after fixation.Therefore we added Annexin V staining of cell membranes, which exacerbated the fluorescence of the membranes and allowed us to see clearly the membrane boundaries in a shorter exposure time.However, your comment suggests us that Annexin V and eGFP have the same green fluorescence, which is indeed not easy to distinguish, and it is also easy to cause confusion in the article.So, we chose Annexin V-PE dye to label the cell membrane and repeated the experiment (see Figure 2D, 4E).And we modified the materials and methods involving adhesion experiments(see line 322).
-In figures 2D and 4E, the TRAF3 OV line appears to have more fluorescence than the control in the representative images.As annexin V is a common marker for apoptosis, is it possible that viability of the OV line is altered even without spore addition, and that the down-regulation of inflammatory cytokines in later figures is simply a consequence of toxicity induced by the robust overexpression of this protein?Quantifying Annexin V fluorescence in the absence of spores and complimenting it with a viability or stress dye (like propidium iodide) would be useful in addressing this concern.

Response:
We agree with the reviewer's comment.We added Supplementary Figure 2 in the manuscript to detect lung epithelial cell viability and cell membrane integrity by MTT assay and LDH release assay.This result showed that overexpression of TRAF3 gene in lung epithelial cells did not affect cell viability changes and cell membrane integrity in the absence of A. fumigatus spores(see line 145-152).Therefore, it can be speculated that the down-regulation of pro-inflammatory cytokine expression is not related to the consequences of toxicity induced by the robust overexpression of the TRAF3 protein (see Supplementary Figure 2A, B).
-In figure 3, the authors thoroughly showed that TRAF3 overexpression attenuated theproduction of key pro-inflammatory cytokines by A549 cells in response to fungal spores.Given the positive correlation between TRAF3 activation and type I interferon, it would be interesting to see if IFN-I was inversely increased in response to TRAF3 overexpression and spore stimulation.
Response: Thank you for pointing this out.Type I IFN is mainly composed of two types, IFN-α and IFN-β.During the experimental process of this study, we also detected the expression of IFN-α and IFN-β, but their expression was very low in A549 cells, and was difficult to be detected.By reviewing the literature, we found that the expression of type I IFN is induced by viral attack.And, IFN-I is mainly produced by fibroblasts and leukocytes.These points can support our qPCR results, so the role played by IFN-I in TRAF3 overexpression and spore stimulation was not considered in this study.References: -Pestka S, Krause CD, Walter MR. Interferons, interferon-like cytokines, and their receptors.Immunol Rev. 2004 Dec;202:8-32. doi: 10.1111/j.0105-2896.2004.00204.x.PMID: 15546383.
-The authors did a nice job looking at signaling pathway activation in figure 5.However, the total levels of these proteins need to be considered as well, as the stoichiometry between activated(phosphorylated)/total protein concentrations is a driving factor in these responses.Quantification (like what was done for the phosphorylated states of these proteins) showing if TRAF3 overexpression impacts total p65, IkBa, ERK and AKT levels should be included to provide justification that overexpression is impacting activation specifically, rather than simply altering the basal levels of these proteins.
Response:We agree with the reviewers' comments.Therefore, we modified the grayscale value analysis in Figure 5B, G.The total levels of p65, IkBa, ERK, and AKT were quantified in Figure 5A, F. The ratio of phosphorylated protein value to total protein value was employed to express the changes in protein activation (see Figure 5B, G).
-In figure 5C, quantification of nuclear translocation would help strengthen the claim that TRAF3 OV inhibits p65 nuclear translocation in response to spores.

Response:
We agree with the reviewers' comments.Therefore, we supplemented Figure 5C, D. Proteins were extracted from the nucleus and cytoplasm, separately, and their p65 protein expression was detected by Western blot to quantify the nuclear displacement of p65 (see Figure 5C, D) to clarify the concerns of the reviewers.
-It would be interesting to see what happens if fungal cells germinate following internalization.As the TRAF3 OV lines have more internalized spores, it is reasonable to assume that these cells will be more damaged during fungal germination, and consequently may increase pro-inflammatory cytokine production and macrophage recruitment later in the infection process.This information could help explain the hypervirulent phenotype observed in transgenic zebrafish as well.
Response: Thank you for pointing this out.We have added Supplementary Figure 2 to the manuscript to verified the effect of TRAF3 overexpression subsequent to the viability and cell damage of A. fumigatus-infected lung epithelial cells.The results showed that TRAF3 did not significantly impact the alteration of A549 cell viability induced by A. fumigatus.Therefore, it can be assumed that TRAF3 inhibition of A. fumigatus-induced pro-inflammatory cytokine production is not related to cell injury(see line 145-152).
The results of the previous study of our team showed that Aspergillus fumigatus started to germinate at 6h when cultured in vitro.When A. fumigatus was co-cultured with human umbilical vein endothelial cells (HUVEC), HUVEC was able to increase the germination time of A. fumigatus spores to 8 h.The first events that occur when A. fumigatus spores infect the host are adhesion and internalization.Combining the literature and the previous research results of our team, we believe that the main events occurring within 6h of A. fumigatus infecting cells in vitro are adhesion and Text Comment -In line 63, the authors state "Some studies have shown that A. fumigatus infects lung epithelial cells in vitro for 6 hours".Is this specifically referring to the spores prior to germination?Clarifying this statement would be helpful.
Response: Thank you for your advice.We have made additions here.It is clarified that when A. fumigatus infects lung epithelial cells in vitro, it first adheres to the cell surface and then internalizes into the cell, with the event that A. fumigatus spores begin to germinate 6 hours after infection(see line 62-64).
Response: We agree that the review raises an important issue.Referring to the reviewer's comments and taking into account the published literature, we have added in the Discussion section of the manuscript additional reasons why the TRAF3 knockout model was not used in this study.TRAF3 knockout mice die in the early stages of life, indicating that this gene has an important biological function in postnatal development and in maintaining normal immune system function.Moreover, there are no agonists or inhibitors available to investigate the TRAF3 gene, which makes the study of the biological function of TRAF3 more difficult.First of all, in this research we found that TRAF3 gene expression was down-regulated after infection of lung epithelial cells with A. fumigatus.Considering also the difficulty of animal model construction and the consistency of in vivo and in vitro experiments.In this study, we chose to construct an in vivo and in vitro model of TRAF3 gene overexpression to in turn demonstrate the role played by TRAF3 gene in the early infection process of A. fumigatus(see line 213-220).

Response:
•Thank you for pointing this out.We have changed the title(see line 103), " FIG1 TRAF3 down-regulation responds to A. fumigatus infection in lung epithelial cells," to read, " FIG1 Lung epithelial cell TRAF3 expression is down-regulated under A. fumigatus infection."If it looks better this way?
•Thank you for your advice.This comment can suggest that non-overlaid black/white images help increase the contrast between different fluorescences.So, we followed your suggestion and tried to change 1D to a black/white image (see

Response:
•Thank you for your affirmation.
•Thank you for your advice.We set the Figure 2D image to black/white, but we do not think the difference is significant (after the image conversion, the contrast of the effect is similar to the comparison image placed above, so we will not show it here).Considering that we applied image J software to count the number of cells and spores ( see Figure 2E ), thus no changes were made to the original image.• Using "-" to represent "normal" TRAF3 expression is misleading as readers are more likely to interpret it as knockdown or knockout.Please use different nomenclature for TRAF3 than +/-to avoid confusion.Especially because "+/-" is used accurately to represent "presence/absence" of spores.

Response:
• Thank you for your affirmation.
•We agree with the reviewers' comments.We chose "Vector" to represent A549 cells transfected with the control vector plasmid and "TRAF3" to represent A549 cells transfected with the TRAF3 overexpression plasmid (see Figure 3).

Response:
•Thank you for your comments.We changed the title of Figure 4 from "Role of TRAF3 in the interaction between lung epithelial cells and macrophages in A. fumigatus infection."to " TRAF3 inhibits macrophage migration and macrophage cytokine production by lung epithelial cells infected with A. fumigatus.".If it looks better this way?
•Thank you for pointing this out.We chose "Vector" to represent A549 cells transfected with the control vector plasmid and "TRAF3" to represent A549 cells transfected with the TRAF3 overexpression plasmid (see Figure 4 ).
• We agree with the reviewers' comments.We chose "Vector" to represent A549 cells transfected with the control vector plasmid and "TRAF3" to represent A549 cells transfected with the TRAF3 overexpression plasmid.Meanwhile, we changed the legend "A549+Spore", "A549-TRAF3+Spore" to "Vector+Spore", "TRAF3+Spore" for for the presence of spores(see Figure 4D ).
•Thanks to the reviewers for their comments.We tried changing the fluorescence image of 4E to black/white to enhance the contrast, but we still think the original image better illustrates the phenomenon(after the image conversion, the contrast of the effect is similar to the comparison image placed above, so we will not show it here).Meanwhile, considering that we applied image J software to count the number of cells and spores, we did not change the original figure(see Figure 4F).• Single channel images should be black and white for contrast.Overlays can be color.
• p-ERK and p-AKT Western (5D) and quantitation (5E) data do not seem to visually agree with each other.Perhaps the blot was overexposed?Is there a better representative image?Was p-ERK and p-AKT normalized to ERK and AKT, respectively, and not GAPDH?These discrepancies between 5D and 5E undermine confident takeaways of the conclusions made from this figure.

Response:
•Thank you to the reviewers comment.We tried to change the fluorescence picture of 5E to black/white to enhance the contrast, but we still think the original picture better illustrates the phenomenon.Furthermore, we added Western blot experiments of protein expression of p65 in the nucleus and cytoplasm (see Figure 5C and D), which supplemented to illustrate that TRAF3 was overexpressed in A549 cells, the accumulation of p65 in the nucleus of A549 cells induced by stimulation of A. fumigatus was significantly reduced.
•We agree with the reviewer's comments.We chose to replace Figure 5D(see Figure 5F) with another set of Western blot images from the duplicate experiments.we also quantified the total levels of p65, IkBa, ERK, and AKT in Figure 5, and the ratio of phosphorylation/total protein concentration was also chosen to indicate the degree of protein activation in the grayscale value analysis( see Figure 5B, F, G ). • Show TRAF3 relative mRNA in zebrafish uninfected with A. fumigatus spores to demonstrate overexpression in vivo.This could be supplemental data.

Response:
• Thank you for your advice.We have changed the title of Figure 6 from "Role of TRAF3 in resistance to A. fumigatus infection in zebrafish." to " TRAF3 overexpression reduces survival and promotes fungal load in vivo in A. fumigatus -infected zebrafish.".If it looks better this way?
•We agree with the reviewer's comments.The mRNA expression of TRAF3 in zebrafish uninfected with A. fumigatus spores is supplemented in Figure 6B to demonstrate the successful construction of transgenic zebrafish overexpressing the TRAF3 gene(see Figure 6B).

Response:
•Thank you for your affirmation.

Figure 4 •
Figure 4 • Update caption title to describe the figure's data and summary takeaway.• Similar comment as in Fig 3 -use different nomenclature other than (-) to indicate normal/control TRAF3 levels.• Fig 4D -please ensure figure graph legend is clear: "A549-TRAF3" suggests at a quick glance a loss of TRAF3 when in fact it is being overexpressed.Consider, for example, "A549+TRAF3" which would still work in the other condition with spores "A549+TRAF3+Spore" • Same image comments as before.

Figure 1 •
Figure 1 • Consider rewording the figure caption: "FIG1 TRAF3 down-regulation responds to A. fumigatus infection in lung epithelial cells".• Make non-overlay images in (1D) black/white for contrast and enhance magnification to clearly illustrate co-localization of DAPI and TRAF3 signals.
Figure II below), and there was little difference in the changes shown with the original image (see Figure I below) for TRAF3 expression.Therefore, we did not change the color scheme of the original image and only supplemented the local zoomed image to show the co-localization of DAPI and TRAF3 signals.Meanwhile, we supplemented Figure1Ewith the average fluorescence intensity calculated using image J software to show the differences in TRAF3 expression (see Figure1E).
FIG. TRAF3 expression and localization in lung epithelial cells.(I) Figure 1D in the original text; (II) TRAF3 fluorescence signals were converted to black/white color matching and merged.

Figure 2 •
Figure 2 • Excellent figure caption title • Same image comments as for Fig.1 -make non-overlay images black/white for contrast and only use color for overlays.Consider also changing the colors (for example, DAPI doesn't have to be dark blue) on overlay for enhanced contrast.

Figure 3 •
Figure 3 • Excellent figure caption title • Using "-" to represent "normal" TRAF3 expression is misleading as readers are more

Figure 4 •
Figure 4 • Update caption title to describe the figure's data and summary takeaway.• Similar comment as in Fig 3 -use different nomenclature other than (-) to indicate normal/control TRAF3 levels.• Fig 4D -please ensure figure graph legend is clear: "A549-TRAF3" suggests at a quick glance a loss of TRAF3 when in fact it is being overexpressed.Consider, for example, "A549+TRAF3" which would still work in the other condition with spores "A549+TRAF3+Spore" • Same image comments as before.

Figure 5
Figure 5• Single channel images should be black and white for contrast.Overlays can be color.•p-ERK and p-AKT Western (5D) and quantitation (5E) data do not seem to visually

Figure 6 •
Figure 6• Update caption title to describe the figure's data and summary takeaway.•Show TRAF3 relative mRNA in zebrafish uninfected with A. fumigatus spores to

Figure 7 •
Figure 7• Excellent schematic that summarizes the paper's data internalization, which is the main research content of our manuscript.As follows: -Zhang W, He D, Wei Y, Shang S, Li D, Wang L. Suppression of Aspergillus fumigatus Germination by Neutrophils Is Enhanced by Endothelial-Derived CSF3 Production.