Lipopolysaccharide-Induced Immunological Tolerance in Monocyte-Derived Dendritic Cells
Round 1
Reviewer 1 Report
Mbongue and colleagues contributed a review about LPS-induced immune tolerance. The topic is interesting. I have several suggestions:
Major:
1. The manuscript content is not well correlated with title. Although the content included extensive review about LPS and LPS related inflammation, the section about immune tolerance should be expanded further to better correspond to the original title.
2. Representative signaling pathways by Figures and Tables about current understanding of LPS tolerance should be added.
3. The mechanisms of LPS tolerance, such as cross tolerance, chromatin remodeling, miRNAs etc should be included in the review.
Author Response
We thank you for your remarks and suggestions.
- “The manuscript content is not well correlated with title. Although the content included extensive review about LPS and LPS related inflammation, the section about immune tolerance should be expanded further to better correspond to the original title”: To this comment, we have added a section (4.1) on Endotoxin Tolerance to better reflect the theme of our manuscript.
- “Representative signaling pathways by Figures and Tables about current understanding of LPS tolerance should be added”: We have removed the initial first figure in the manuscript and updated the second one with recent knowledge on endotoxin tolerance pathways.
- “The mechanisms of LPS tolerance, such as cross tolerance, chromatin remodeling, miRNAs etc should be included in the review”: In Section 4.1 we have discussed cross-tolerance, chromatin remodeling and miRNA impact on endotoxin tolerance.
Reviewer 2 Report
The review by Mbongue et al is overall well written and a comprehensive overview of LPS and its role in immune cell activation. However, the review is not focused, and several areas need clarification.
My main comment is that the supposed focus of the paper, the effects of LPS on immune tolerance of DCs (based on the title), is lost. The potential for LPS to induce DC tolerance is only briefly discussed towards the very end, and this discussion needs significant clarification. This section more describes cholera toxin and it is not clear how LPS fits in. Also the authors list TGFb and IL-10 as pro-inflammatory cytokines, when they are known to be anti-inflammatory. There is also a statement that tolerogenic DCs inhibit proliferation of anti-inflammatory T cells, which is either incorrect or confusing. In section 5.2.3 the authors state that LPS has been shown to induce a pro-inflammatory phenotype in DC, with up-regulation of IL-10 and IDO, again is this supposed to describe a tolerogenic state? The focus of the review based on the title needs significant editing.
Based on the title, can the authors go into more detail about dendritic cell subsets. Why is it important to point out tolerance in monocyte-derived dendritic cells?
Another overarching comment is that the review seems to be written by several people and put together without going through the manuscript for consistency. The abbreviation for lipopolysaccharide (LPS) is given numerous times, so is TLR4 and an explanation for TLR4 is given numerous times as well.
The sections on LPS structure and especially biosynthesis seem not necessary for the focus of this review.
In the introduction, the sentence “Said et al showed that LPS upregulated the expression of the death receptor PD-1 on monocytes and upon engaging the cognate ligand PD-L1 increases IL-10 expression and inhibition of CD4-Tecll expansion” is out of place and needs to be moved to somewhere more appropriate, or removed completely.
Author Response
We thank the reviewer and agree with this criticism. To address this comment we have revised the manuscript with an expanded section on immune tolerance.
“My main comment is that the supposed focus of the paper, the effects of LPS on immune tolerance of DCs (based on the title), is lost. The potential for LPS to induce DC tolerance is only briefly discussed towards the very end, and this discussion needs significant clarification. This section more describes cholera toxin and it is not clear how LPS fits in. Also the authors list TGFb and IL-10 as pro-inflammatory cytokines, when they are known to be anti-inflammatory. There is also a statement that tolerogenic DCs inhibit proliferation of anti-inflammatory T cells, which is either incorrect or confusing. In section 5.2.3 the authors state that LPS has been shown to induce a pro-inflammatory phenotype in DC, with up-regulation of IL-10 and IDO, again is this supposed to describe a tolerogenic state? The focus of the review based on the title needs significant editing.” We the authors agree and therefore added section 4.1 that extensively discusses the recent knowledge on endotoxin tolerance and the role SHIP-1, IRAK-M and miRNAs play in it. To the question of how LPS fits into the effects of CTB-INS immunotolerance, we have extensive evidence of immune suppression by CTB-INS both in vivo and in vitro. The CTB-INS fusion gene was cloned into the E. coli PBR-322 expression vector and the amplified plasmid into the E. coli HB101 strain. The amplification of the CTB-INS protein in that E. coli strain was found to contain significant amounts of endotoxin as detailed in Kim et al. Our further investigation identified that an immunosuppressive enzyme named indoleamine 2, 3 dioxygenase (IDO) was upregulated in human monocyte-derived DCs following culture with CTB-INS which was directly related to the presence of LPS. Taken together these results led us to believe that the presence of endotoxin may further promote tolerance within DCs in addition to any direct immunosuppressive effects of CTB-INS. This last point is important as the expression of CTB-INS within plants which would not generate LPS, was studied in vitro and in experimental animals and found to be immunosuppressive. This was detailed throughout the review. In addition to clarifying this point, we have fixed all the other errors pointed out by the reviewer.
- “Based on the title, can the authors go into more detail about dendritic cell subsets. Why is it important to point out tolerance in monocyte-derived dendritic cells?”
The authors understand and recognize the existence of subsets of DCs. In our study, monocyte-derived dendritic cells (MoDCs) were prepared from freshly collected human peripheral blood cells isolated from aphaeresis filter cones obtained from a local blood bank. In the protocol outlined in Mbongue et al. (2016) we cultured CD14+ monocytes with GMCSF and IL-10 until their differentiation into CD11c+ dendritic cells prior to treatment with CTB-INS in vitro. Monocyte-derived dendritic cells (Mo-DCs) are a distinct subset of DCs involved in inflammation and infection and originate from monocytes during circulatory stimulation, and their activation and function are significant in autoimmune diseases. It is in this specific DC subset that the immunosuppressive effect of CTB-INS was observed. The authors have not observed this effect in human macrophages and have not tested it in other DC subsets such as plasmacytoid DCs (pDC) or conventional DCs (cDC). Lastly, there is an important future clinical objective in that isolating monocytes from patients and then converting them to DCs and treating them in vitro for clinical immunosuppression would be highly efficient.
- “Another overarching comment is that the review seems to be written by several people and put together without going through the manuscript for consistency. The abbreviation for lipopolysaccharide (LPS) is given numerous times, so is TLR4 and an explanation for TLR4 is given numerous times as well.”
We the authors agree on the issue of redundancy and have addressed it thoroughly throughout the manuscript.
- The sections on LPS structure and especially biosynthesis seem not necessary for the focus of this review.
We have agreed to this comment and removed entirely the section on LPS structure.
- “In the introduction, the sentence, “Said et al showed that LPS upregulated the expression of the death receptor PD-1 on monocytes and upon engaging the cognate ligand PD-L1 increases IL-10 expression and inhibition of CD4-Tecll expansion” is out of place and needs to be moved to somewhere more appropriate or removed completely.”
The authors also agreed with this remark, and we have removed the sentence from the end of the introduction.
Reviewer 3 Report
The authors addressed a nice and updated review of Lipopolysaccharide-Induced Immunological Tolerance in Monocyte-derived Dendritic Cells.
Some grammar errors need to be checked, words in italics (in vivo), and the format must be reviewed.
Author Response
We thank you for the positive comment on our review.
“Some grammar errors need to be checked, words in italics (in vivo), and the format must be reviewed.”
The authors have reviewed the manuscript for grammar errors and formatting issues.
Reviewer 4 Report
The article is a well-written review of immunological mechanisms of tolerance induced by LPS in Mo-DCs; however some aspects are still missing that could explain more about the LPS function on tolerance:
a) Activation of DC is described, but a mention of costimulatory molecules is missing. For example, how do costimulatory molecules participate in tolerance after LPS stimulation?
b) Important aspects of immune suppression induced by LPS are discussed in section 5, but what about the regulation mechanisms, alternative ways, or counterbalance mechanisms? Why is it so important to understand these phenomena?
Minor observations
Check abbreviations all along the paper, for example "E. E. coli" in 5.2.1; or add meaning, for example "Mal" in figure 2
Author Response
“The article is a well-written review of immunological mechanisms of tolerance induced by LPS in Mo-DCs; however, some aspects are still missing that could explain more about the LPS function on tolerance:
- Activation of DC is described, but a mention of costimulatory molecules is missing. For example, how do costimulatory molecules participate in tolerance after LPS stimulation?”
Thank you for the compliment and you have posed an important and relevant question. To address your question, we have included a discussion on the matter in section “4.1 Endotoxin Tolerance”.
- Important aspects of immune suppression induced by LPS are discussed in section 5, but what about the regulation mechanisms, alternative ways, or counterbalance mechanisms? Why is it so important to understand these phenomena?
We thank the reviewer for this important insight and question. As the reviewer is aware, LPS as endotoxin is a major contributor to the adverse effects of sepsis as an inflammatory agent. However, as suggested by the question there is an LPS tolerance balancing the inflammation and driven in part by immunosuppression which can have devastating clinical effects. The primary focus of this review paper was to discuss the potential role LPS tolerance plays in the immunosuppressive effects of the CTB-INS fusion protein in monocyte-derived DCs. However, our findings of the role of IDO in DC biology after exposure to LPS and the antigen suggest a novel pathway for LPS immunosuppression in conditions such as sepsis. To keep with the theme of the review we have expanded on the topic of the immunosuppressive effects of endotoxin tolerance.
Round 2
Reviewer 2 Report
The authors have sufficiently addressed my prior concerns.
Reviewer 4 Report
All observations were addressed in the new manuscript version
