Immunomodulatory Responses of Two Synthetic Peptides against Salmonella Typhimurium Infection

Round 1

Reviewer 1 Report

This is an interesting research and the methods for tackling the research objectives are appropriate. However several points regarding the results need to be considered: Introduction: -Background knowledge of salmonella intestinal infection should be given, in order to reflect the choice of disease model and the matrices of the analyses are sound. Results: -For the cell viability experiments, can you please explain why such different concentration ranges were used for each peptide? Pin2[G]: 0.03-10 g/mL, whereas FA1: 1.17-150 g/mL. Is it based on the literature? (if so, appropriate papers should be cited). -The sensitivity of BMDM to the peptides didn’t show a clear concentration-dependent relationship. The cells were less viable at lower concentration (0.31 and 0.63 g/mL for Pin2[G]; 4.69 g/mL for FA1) but somehow survived better at higher concentrations. Please explain. -The claim at the conclusion of the cell viability result section “the peptide could decrease the macrophages population, but without significantly affecting their viability” is not valid as the experiments only showed viability results. I’m not sure what “decrease cell population” was referring to. -For the phagocytosis experiments, same range of concentrations were used for both peptide in both macrophage cell types. However, given the different viability data showed in Section 2.1, the numbers of viable macrophages weren’t taken into account for this set of experiment. Did the author normalize the CFU count by the number (or percentage) of viable cells? -Caption of Figure 3: “Peptides do not change cytokine expression profile in un-stimulated mouse macrophage” where as in Line 130 it says the concentrations of MCP-1 and TNF were decreased in the presence of both peptides before infection and the results graphs for MCP-1 and TNF also indicated the changes were significant. -For the cytokine profile experiments, the most dramatic changes between pre- and post-infection were seen in MCP-1 and TNF. However the no peptide negative control also showed a vast difference in the release of these two cytokine. MCP-1 showed a more than 5-fold reduction after challenge, and TNF showed an even higher magnitude reduction, from ~1800 pg/mL pre-infection to less than 100 pg/mL post-challenge. This different wasn’t seen in the plasma cytokine concentrations in the in vivo mouse experiments (Figure 6), making us questioning the validity of the cell line model. -For the cytokine profile analyses, a smaller range of peptide concentrations was used. However the authors need to elaborate the rationale of choosing these concentration ranges, because the concentrations chosen for Pin2[G[ (1.25-10 g/mL) did not show any effect on macrophage cell viability or phagocytosis (from Figures 1 and 2); whereas the concentration range chosen for FA1 (18.75-150 g/mL) did have effect on both cell viability and phagocytosis. This gives the comparison between the two peptides a very different standpoint. -Based on the manufacturer’s information sheet for the cytokine CBA kits (BD cat. #552364) used in this research, for IL6 and INF, the low values in the results of this research is very close to the detection limit, and almost out of the linear range. Experiments for these two particular cytokines may need adjustment, ie the samples need to be concentrated or enriched. -For the murine infection model, a very small sample size (n=3 for each treatment) was used. Please provide statistics for this sample size calculation, to show that the results can reflect significance for the purpose of the tests. The article provides some useful information on the topic; however the language needs to be revised extensively. Some paragraphs seem to have omission, typos, and incorrect grammar. Suggested revisions, for examples, are: Line 18: add “(BMDM)” after “bone marrow derived macrophages”. Revise sentence in Lines 23-25. Line 29: revised as “in” gastric infections by “S. typhimurium“(and should be in italic). Line 78: typo: “HPDs” should be “HDPs”. Line 79: revised as “we have shown at such concentrations, Pin2[G] and FA1 caused the death of…” Line 84: revised as “of BMDM almost to half”. Line 108: revised as “On the other hand, FA1 also stimulated phagocytosis but at higher concentrations.” Line 127: “the cytokines present in the macrophages supernatant after 24-hour incubation with different concentrations of peptides, followed by challenge with S. typhimurium 30 minutes later.” And throughout the article, bacteria species names should be in italic, and "IFN" should be "IFN-gamma" or "IFN-g" Please also supply better resolution images for Figures 1 and 2.

Author Response

Comments and Suggestions for Authors

This is an interesting research and the methods for tackling the research objectives are appropriate. However, several points regarding the results need to be considered:

 

Introduction: -Background knowledge of salmonella intestinal infection should be given, in order to reflect the choice of disease model and the matrices of the analyses are sound.

Answer: We’ve included some more information in the Introduction section, lines from 45 to 60.

 

Results:

-For the cell viability experiments,

-can you please explain why such different concentration ranges were used for each peptide? Pin2[G]: 0.03-10 g/mL, whereas FA1: 1.17-150 g/mL. Is it based on the literature? (if so, appropriate papers should be cited).

Answer: Yes, we have previously found the cytotoxic effect of both peptides; therefore, our rational to use such concentrations were based in those reported results. We included a sentence (lines 356-358) to be more explicit on this issue: “Such peptide concentrations were used based on a previous report; that is, Pin2 [G] and FA1 concentrations higher than 10 and 150 µg/mL, respectively, are toxic for RAW 264 cells [14].”

-The sensitivity of BMDM to the peptides didn’t show a clear concentration-dependent relationship. The cells were less viable at lower concentration (0.31 and 0.63 g/mL for Pin2[G]; 4.69 g/mL for FA1) but somehow survived better at higher concentrations. Please explain.

Answer: Such results were also unexpected to us, and perhaps more independent experiments will help to visualize how these peptides affects cell viability. We added a paragraph (lines 244 to 252) to discuss such results: “An interesting observation is that macrophage toxicity was not a concentration-dependent event, which is the classic mechanism of action of AMPs. Therefore, some other biochemical mechanisms may be hampered at low concentrations of such peptides; for instance, peptide interaction with some cell targets that affect viability, or even interference with DNA replication, this effect of sub-inhibitory concentrations of peptides has been previously reported [36, 37]. To explain such results some other experiments are required concerning the interaction of Pin2 [G] and FA1 with proteins or genetic elements of immune cells.”

-The claim at the conclusion of the cell viability result section “the peptide could decrease the macrophages population, but without significantly affecting their viability” is not valid as the experiments only showed viability results. I’m not sure what “decrease cell population” was referring to.

Answer: You’re correct, we apologize for such mistake, we’ve rewritten such sentence to say just “decrease macrophage viability” because our measure isn’t a direct determination of cell population.

-For the phagocytosis experiments,

-same range of concentrations were used for both peptide in both macrophage cell types. However, given the different viability data showed in Section 2.1, the numbers of viable macrophages weren’t taken into account for this set of experiment. Did the author normalize the CFU count by the number (or percentage) of viable cells?

Answer: Yes, macrophages were normalized at a density of 1x10⁵ cells/well.

-Caption of Figure 3:

“Peptides do not change cytokine expression profile in un-stimulated mouse macrophage” where as in Line 130 it says the concentrations of MCP-1 and TNF were decreased in the presence of both peptides before infection and the results graphs for MCP-1 and TNF also indicated the changes were significant.

Answer: Thank you again. We’ve changed the name of such figure to “Peptides decreases MCP-1 and TNF expression in unstimulated mouse macrophages Raw 264.7”

-For the cytokine profile experiments,

the most dramatic changes between pre- and post-infection were seen in MCP-1 and TNF. However, the no peptide negative control also showed a vast difference in the release of these two cytokines. MCP-1 showed a more than 5-fold reduction after challenge, and TNF showed an even higher magnitude reduction, from ~1800 pg/mL pre-infection to less than 100 pg/mL post-challenge. This different wasn’t seen in the plasma cytokine concentrations in the in vivo mouse experiments (Figure 6), making us questioning the validity of the cell line model.

Answer: After double checking, databases and analysis, we realize that the results between pre- and post-infection concerning MCP1 and TNF were plotted incorrectly, that is, the graphics were exchanged. An error that is extremely regrettable. We really appreciate the time and effort that you have dedicated to providing your valuable feedback, in this way it was possible to detect this particular mistake and make the corrections.

-For the cytokine profile analyses,

-a smaller range of peptide concentrations was used. However the authors need to elaborate the rationale of choosing these concentration ranges, because the concentrations chosen for Pin2[G[ (1.25-10 mg/mL) did not show any effect on macrophage cell viability or phagocytosis (from Figures 1 and 2); whereas the concentration range chosen for FA1 (18.75-150 mg/mL) did have effect on both cell viability and phagocytosis. This gives the comparison between the two peptides a very different standpoint.

Answer: The reviewer is correct. Our rationale was to test the higher concentrations of Pin2[G] and FA1 to look for cytokine release without jeopardizing totally the cell viability. That is, most of the concentrations of Pin2[G] did not affect macrophage viability; therefore, the cytokine release is because a stimulatory effect rather than a cell disruption effect. In the case of FA1, the larger concentrations (75-150 mg/mL) in Raw 264.7 cells had a viability effect, but at the concentrations of 18.75-37.5 mg/mL had not. Therefore, the observed cytokine release of those that are constantly produced in Raw 264.7 cells and treated at larger concentrations of FA1 may be overestimated; however, there were not significant values at such concentrations.

-Based on the manufacturer’s information sheet for the cytokine CBA kits (BD cat. #552364) used in this research, for IL6 and INF, the low values in the results of this research is very close to the detection limit, and almost out of the linear range. Experiments for these two particular cytokines may need adjustment, ie the samples need to be concentrated or enriched.

Answer: We agree; however, we revised again the standard curves to emphasize this point. Based on the manufacturer’s information (instructions sheet for the cytokine CBA kits, BD cat. #552364), we have applied the 4-parameter curve fit option, in order to extrapolate values for sample intensities that did not fit into the limits of the standard curve. For this particular analysis, we used GraphPad – Prism 9 and the FlowJo software tools. Using this strategy, it was possible to detect and quantify values with MFI 3.3 or 5 pg/mL for IL-6 and MFI 5.4 or 2.5 pg/mL for IFN.

-For the murine infection model,

-a very small sample size (n=3 for each treatment) was used. Please provide statistics for this sample size calculation, to show that the results can reflect significance for the purpose of the tests.

Answer: Thank you again, the statistical results are from 3 independent experiments including 3 animals in each group (3 x 3), which is a total sample of 9 individuals for each treatment. So, the description of the figures was re-written: “The mean ± standard deviation, of three independent experiments with three individuals in each group, is shown”. The statistics has taking in account the 3 x 3 plot of each treatment.

The article provides some useful information on the topic; however, the language needs to be revised extensively.

Some paragraphs seem to have omission, typos, and incorrect grammar.

Suggested revisions, for examples, are:

Line 18: add “(BMDM)” after “bone marrow derived macrophages”.

Revise sentence in Lines 23-25.

Line 29: revised as “in” gastric infections by “S. typhimurium“(and should be in italic).

 

Answer: We’ve kept S. Typhimurium since “Typhimurium” is a serovar of Salmonella enterica, A sentence was included (lines 39 to 41). In addition, according to the nomenclature of the American Type Culture Collection, S. Typhimurium is correct.

 

Line 78: typo: “HPDs” should be “HDPs”.

Line 79: revised as “we have shown at such concentrations, Pin2[G] and FA1 caused the death of…”

Line 84: revised as “of BMDM almost to half”.

Line 108: revised as “On the other hand, FA1 also stimulated phagocytosis but at higher concentrations.”

Line 127: “the cytokines present in the macrophages supernatant after 24-hour incubation with different concentrations of peptides, followed by challenge with S. typhimurium 30 minutes later.”

 

And throughout the article, bacteria species names should be in italic, and "IFN" should be "IFN-gamma" or "IFN-g" Please also supply better resolution images for Figures 1 and 2.

 

Answer: Corrections were made, thank you very much. Also, figures are included now with a better resolution.

 

Reviewer 2 Report

Corzo and co-workers have presented an interesting study regarding the antimicrobial and immunomodulatory properties of two synthetic peptides Pin2[G] and FA1 against Salmonella enterica serovar Typhimurium using in vitro and in vivo assays. The study design is sound, the methodologies applied were adequate, the results are clearly presented, and the conclusions are fully supported by the results. Generally speaking, the manuscript is well written and is appropriate for publication in the special issue of Molecules. However, before it can be accepted, there are still some minor points that need to be addressed by the authors.

1. In the Introduction section, although the authors mentioned that both  Pin2[G] and FA1 showed promising antimicrobial activity against several reference strains and clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa at in vitro level and in in vivo models of skin infection in their previous reports, they need to provide more explanation why they focus on the  therapeutic ability of these two AMPs against S. Typhimurium infection.
2.  Line 61, "HDOs", line 78, "HPDs", these are typos that need to be corrected to "HDPs".
3.  In Figures 1 to 6, the resolution of the text and numbers are not good enough for readers using either on-line or printed version of the PDF file. The authors should improve the resolution of these figures.
4. According to the results, although both peptides do not completely eliminate the gastric infection, they do decrease bacterial load in feces at the same time that they modulate the immune response. As the authors mentioned in the Discussion section that a modification in the dosage or its use in conjunction with antibiotics will allow to develop better conditions for treatment. Since these peptides exhibit different immunomodulatory functions, would that be possible to perform combination usage of Pin2[G] and FA1 on S. Typhimurium infections and see if there's synergistic effects with certain dosage ratio applied.

Author Response

Corzo and co-workers have presented an interesting study regarding the antimicrobial and immunomodulatory properties of two synthetic peptides Pin2[G] and FA1 against Salmonella enterica serovar Typhimurium using in vitro and in vivo assays. The study design is sound, the methodologies applied were adequate, the results are clearly presented, and the conclusions are fully supported by the results. Generally speaking, the manuscript is well written and is appropriate for publication in the special issue of Molecules. However, before it can be accepted, there are still some minor points that need to be addressed by the authors.

1. In the Introduction section, although the authors mentioned that both Pin2[G] and FA1 showed promising antimicrobial activity against several reference strains and clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa at in vitro level and in in vivo models of skin infection in their previous reports, they need to provide more explanation why they focus on the therapeutic ability of these two AMPs against S. Typhimurium infection.

 

Answer: Thanks for your comment, we’ve added a paragraph with a brief description of our previous experiments: “At the same time, it was observed that both peptides have certain effect on the release of MCP-1 and IL-6 by RAW264.7 and HBE macrophages [14]. Furthermore, in a model of gastrointestinal infection by S. Typhimurium ATCC 14028 was observed that the administration of Pin2[G] intravenously reduces bacterial presence in feces, but does not completely inhibit it. Therefore, our interest arises to understand the role of such peptides as immunomodulators in this type of infections.

 

 

1. Line 61, "HDOs", line 78, "HPDs", these are typos that need to be corrected to "HDPs".

 

Answer: They were corrected, thank you.

 

1. In Figures 1 to 6, the resolution of the text and numbers are not good enough for readers using either on-line or printed version of the PDF file. The authors should improve the resolution of these figures.

 

Answer: They were corrected, thank you.

 

1. According to the results, although both peptides do not completely eliminate the gastric infection, they do decrease bacterial load in feces at the same time that they modulate the immune response. As the authors mentioned in the Discussion section that a modification in the dosage or its use in conjunction with antibiotics will allow to develop better conditions for treatment. Since these peptides exhibit different immunomodulatory functions, would that be possible to perform combination usage of Pin2[G] and FA1 on S. Typhimurium infections and see if there's synergistic effects with certain dosage ratio applied.

 

 

Answer: Currently, we’re working on the design of treatments based on the joint administration of FA1 and Pin2[G] or ceftriaxone with both antimicrobials, although we have observed interesting results at the level of cytokine expression profiles, we still need to verify this information to eventually publish it.

Round 2

Reviewer 1 Report

Thank you for addressing all the questions raised and updating the parts of the manuscript accordingly.