Intermittent Exposure of Hypercapnia Suppresses Allograft Rejection via Induction of Treg Differentiation and Inhibition of Neutrophil Accumulation
, Yi-Jen Peng 3,4, Shih-En Tang 5,6, Kun-Lun Huang 1,6, Shi-Jye Chu 7, Shu-Yu Wu 5,* and Chia-Pi Cheng 8,*Round 1
Reviewer 1 Report
The mechanistic experimental results are very weak and the conclusions are over amplified. The induction of Treg upon HCA exposure is not convincing.
1- Figure 7 has unspecific staining.
2- western blot data to show the Treg is very weak.
3- Figure 8 states that micrograph is representative of lung tissue?
Author Response
Response to Reviewer 1 Comments
Point 1- Figure 7 has unspecific staining.
Response: We thank reviewer for this comment and agree with you. We can’t exclude that unspecific staining was display in Figure 7. However, we have already redid this stain by using monoclonal antibodies and reducing antibodies concentration to decrease the nonspecific binding. The staining sections were performed at the same time and the unspecific staining could be as background level between each section. The revised data as shown in new Fig. 7.
Point 2- western blot data to show the Treg is very weak.
Response: We thank reviewer for this comment. We knew the quantitative experiments of Treg related proteins were not so dramatical increase in vivo. But we saw the tendency. That’s the reason why we did the in vitro further examination to directly detect HCA function on Treg cell differentiation as shown in Figure 8.
Point 3- Figure 8 states that micrograph is representative of lung tissue?
Response: We thank reviewer for reminding us of our writing error. We have revised the sentence and corrected the error as shown in Figure 9 (p.11, lanes 271 in simple markup mode).
Reviewer 2 Report
Title Intermittent Exposure of Hypercapnia Suppresses Allograft Rejection via Induction of 2
Treg Differentiation and Inhibition of Neutrophil Accumulation
Authored by: Y-S Tzeng et al.
The paper concerns effects of intermittent hypercapnia upon rejection of skin allografts and concomitant inflammation markers in murine model. The present study involved skin transplantation experiments from BALB/c to C57BL/6 mice. Hypercapnia levels were controlled by measuring target pH and PCO2 values. The results obtained clearly show a decreased inflammatory response in CO2-treated animals which correlated with attenuation of necrotic lesions. Suppressed inflammation is confirmed by decreased serum Th1 cytokines, and lower CD8+ cell contents in lymphoid tissues. This may be a pre-requisite for the Treg activation. Reduction in local Ly6g+ neutrophils also suggests protective effects of CO2 conditioning. The Discussion is well written, concerning the main findings shown in Results which are clearly presented.
There are only few remarks:
Materials and methods:
Regimen of CO2 administration is critical to the clinical outcomes. Therefore, timing of the 5% CO2 exposure should be clearly specified (p.2, lanes 96-98), i.e, days of CO2 conditioning post-transplant (D+1, +2 etc.?). Was a pre-transplant conditioning used?
The manuscript needs only slight language editing.
Conclusion.
The article concerns an important issue, is well performed as a well-designed experimental study with proper analysis of relevant immune populations and humoral factors and deserves publication after minor revisions according to the reviewer’s remarks.
The article can be accepted after minor revisions.
Author Response
Response to Reviewer 2 Comments
Point 1: Materials and methods:
Regimen of CO2 administration is critical to the clinical outcomes. Therefore, timing of the 5% CO2 exposure should be clearly specified (p.2, lanes 96-98), i.e, days of CO2 conditioning post-transplant (D+1, +2 etc.?). Was a pre-transplant conditioning used?
Response: We thank reviewer for this suggestion. In this study, we used a post-transplant exposure condition. The first exposure time to HCA was on day 0 (transplant day), mice were exposed to 5% CO2 in air for one hour after skin transplantation. We have added the exposure time into the revised manuscript as shown in materials and methods 2.3. lanes 97 (simple markup mode).
Point 2: The manuscript needs only slight language editing.
Response: We thank reviewer for this suggestion. We have found an English expertise to improve our language editing.
Reviewer 3 Report
The authors written this piece rather well and in a coherent structure and have been able to address all the main questions. This paper is clearly written and well organized. The introduction and background are reasonable given the premise of the paper. Figures are comprehensive and helpful. The authors showed that hypercapnia intermittent exposure can effectively suppress skin rejection by stimulating Tregs cell generation and suppressing immune reaction.
Minor comments:
- I would be nice in the figure 3 to see sample distribution in the histogram plots, to better appreciate the heterogeneity in the different groups.
- What about the deep phenotype of infiltrating T reg cells are they effector Tregs or Naïve Tregs? We know that tissue infiltrating T cells contained a higher frequency of effector Tregs, which are defined as FOXP3hi and CD45RA−, terminally differentiated, and most suppressive. Naïve Treg cells are FOXP3lo and CD45RA+ and predominantly expressed C-C chemokine receptor 4 (CCR4) in both tissues infiltrate and peripheral blood. And the line with what about circulating immune cells in the blood?
- The phenotype of induced T reg cells in the in vitro experiment could be better investigate (effector, naïve or exhausted Treg cells). The representative FACS plot is not as clear cut.
- Be careful in the figure annotation reference, paragraph 3.3; line 231 refers to (Fig.5B, F and not E) the same, line 233 refers to (Fig5 D, H) … etc.
- The diagram of HCA on regulation of Innate and adaptive immune cells in allograft rejection, Figure 10 is clear and summarize well the paper but it is not referenced anywhere in the text.
Author Response
Response to Reviewer 3 Comments
Point 1: I would be nice in the figure 3 to see sample distribution in the histogram plots, to better appreciate the heterogeneity in the different groups.
Response: We thank reviewer for this suggestion. We have revised the figure 3 display by sample distribution.
Point 2: What about the deep phenotype of infiltrating T reg cells are they effector Tregs or Naïve Tregs? We know that tissue infiltrating T cells contained a higher frequency of effector Tregs, which are defined as FOXP3hi and CD45RA−, terminally differentiated, and most suppressive. Naïve Treg cells are FOXP3lo and CD45RA+ and predominantly expressed C-C chemokine receptor 4 (CCR4) in both tissues infiltrate and peripheral blood. And the line with what about circulating immune cells in the blood?
Response: We thank reviewer for this comment and suggestion. We stained Treg cells by CD4 and FOXP3 based on two reasons. First, previous studies have reported that IL-2 therapy would induce CD4 FOXP3 specific Treg cell expansion [1]. Second, low-dose IL-2 therapy has been used in many clinic patients, like GVHD, type 1 DM, and hepatitis [2]. Thus, our priority was checking these two markers first and hope our study quickly used in clinic. Your opinions were a very interesting point and need to further clarify, we may add this section in our future works.
Point 3: The phenotype of induced T reg cells in the in vitro experiment could be better investigate (effector, naïve or exhausted Treg cells). The representative FACS plot is not as clear cut.
Response: We thank reviewer for this suggestion. We revised the figure by using density blot with multi color display to increase the difference between groups.
Point 4: Be careful in the figure annotation reference, paragraph 3.3; line 231 refers to (Fig.5B, F and not E) the same, line 233 refers to (Fig5 D, H) … etc.
Response: We thank reviewer for reminding us of our errors. We apologize to let reviewer confused with these results. We have corrected these errors as shown in page 7, lanes 210 and 212 in simple markup mode.
Point 5: The diagram of HCA on regulation of Innate and adaptive immune cells in allograft rejection, Figure 10 is clear and summarize well the paper but it is not referenced anywhere in the text.
Response: We thank reviewer for this suggestion. We have address Figure 10 as the reference in the conclusion part.
- Treg-mediated prolonged survival of skin allografts without immunosuppression. Pilat N, Wiletel M, Weijler AM, Steiner R, Mahr B, Warren J, Corpuz TM, Wekerle T, Webster KE, Sprent J. Proc Natl Acad Sci U S A. 2019 Jul 2;116(27):13508-13516.
- Low-Dose IL-2 Therapy in Autoimmune and Rheumatic Diseases. Graßhoff H, Comdühr S, Monne LR, Müller A, Lamprecht P, Riemekasten G, Humrich JY. Front Immunol. 2021 Apr 1;12:648408.
