Polymerization of misfolded Z alpha-1 antitrypsin protein lowers CX3CR1 expression in human PBMCs

Thank you for submitting your article "Polymerization of misfolded protein lowers CX3CR1 expression in human PBMCs" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen Satyajit Rath as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another, and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

The reviewers agree that the implications of your work are broad and carry clinical significance, although there are major concerns over the lack of sufficient molecular level understanding of the regulation of CX3CR1's cell surface expression by Z-AAT polymers, for consideration for publication in eLife. Considering these concerns of the reviewers, we can only consider the manuscript further after a major revision if you sufficiently address all the concerns as outlined below.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Summary:

The manuscript by Tumpara S. et al. shows that polymers of mutated α-1 antitrypsin (AAT) affect expression and internalization of CX3CR1, presenting a novel example of CX3CR1 regulation by misfolded proteins and what could be a novel immunomodulatory function of protein α-1 antitrypsin. Using human PBMCs and plasma purified AAT in its wild type monomeric and mutated polymeric forms, authors show that Z AAT polymers reduce CX3CR1 expression and its surface expression. As CX3CR1 has been previously found to be regulated by Tau and amyloid-β, this finding poses a question if other conformationally altered extracellular proteins could have the same property. It also opens a question how this process is affected by inflammatory conditions and what are its implications for individuals with Z AAT. In addition to the novel mechanism of CX3CR1 regulation, these findings could be of relevance for better understanding of molecular pathophysiology associated with AAT deficiency (AATD) adding a potentially important step to the mechanism of AATD pathology. Furthermore, these findings could also have relevance in other conditions in which CX3CR1 axis and AAT have been implicated (e.g. cancer).

Essential revisions:

1. The protein level of CX3XR1 in total cell lysate is high although the gene expression is significantly low in AATD patients or Z-AAT treated cells which has been explained by showing Z-AAT mediated CX3XR1's association with lipid rafts. How specific is this effect? Would any oligomeric protein which can bind to CX3CR1, have similar effect? What is the effect with treatment with monomeric Z-AAT on CX3CR1 gene expression and protein level?

2. There is no explanation provided for decreased expression CX3CR1 in AATD patients or after in vitro polymeric Z-AAT treatment except for indirect connection with increased CD14 expression (CD14++ cells are known to decrease CX3CR1 expression). What is the possible mechanism of such drastic decrease in expression level of CX3CR1?

3. Are there any changes in CX3CR1 expression after fractaline challenge of the PBMCs treated with polymeric Z-AAT?

4. Is there any change in CX3CR1 expression and cell surface expression in cells having intracellular Z-AAT polymers?

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Polymerization of misfolded Z α-1antitrypin protein lowers CX3CR1 expression in human PBMCs" for further consideration by eLife. Your revised article has been reviewed by two reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Satyajit Rath as the Senior Editor.

Although both the reviewers have agreed that the manuscript has been improved by adding further experimental evidence for reduced cell surface expression of CX3CR1 by ZZ-AAT polymers and additional experiments, there are some issues, as outlined below, which need to be addressed before a final decision can be made.

1. The explanation for significant decrease in mRNA expression levels of CX3CR1 in patient PBMCs by ZZ-AAT polymers is not properly supported experimentally. At the very least, an acknowledgement and a plausible explanation of such an important observation is essential.

2. A discussion (supported by experiments if possible) on the fate of internalized CX3CR1 upon removal of ZZ-AAT polymers from surrounding media should be included to increase the impact of the study.

3. The submission is being considered as a short report. It is critical to maintain the appropriate format (such as limiting the total number of main display items within )5, in the revision.

The detailed recommendations from both reviewers are being provided below for ease of response.

Reviewer #1:

In the revised version of manuscript 13-11-2020-RA-eLife-64881R1, Tumpara et al. has successfully addressed the observation of decreased cell surface expression of CX3CR1 on PBMCs in patients of α-1 antitrypsin deficiency (AATD) with circulating polymeric forms of AAT (Z-AAT) protein. They have shown the exclusive role of polymeric ZZ-AAT in CX3CR1 internalization in comparison to monomeric form of the protein or the CX3CL1. The authors mentioned that this finding is already known for other aggregated proteins like Tau, so this finding is not sufficiently novel. Importantly, the most intriguing finding of significant decrease in mRNA level expression of CX3CR1 is not explained except for an indirect correlation with increased CD14 expression by the ZZ-AATD treated cells. This finding needs further explanation. Authors have only explained about reduction of cell surface expression of CX3CR1 which is not satisfactory to explain the decreased mRNA level expression of CX3CR1 with polymeric ZZ-AATD exclusively and its implication in patients of AATD. To be acceptable for publication, this important observation needs to be explained and its implication in patients of AATD needs to be discussed in detail.

Reviewer #3:

Manuscript provides data showing that Z AAT induces reduced CX3CR1 gene expression and protein surface expression while increasing intracellular levels of the protein and new data provided by the authors show that the effect on CX3CR1 expression is executed specifically by Z AAT polymer and not by its monomeric form or polymeric variant of WT protein. Experiments including CXCL1 were added and new colocalization experiment provides an explanation for increased amount of CX3CR1 in cell lysates treated with Z AAT and I believe the manuscript is suitable for publication. It would be interesting to know if upon removal of ZAAT from surrounding media CX3CR1 could be recycled back to the surface or is the interaction more permanent (ZAAT induces increase in intracellular CXCR1 compared to CX3CL1), however that would be beyond the scope of this manuscript. Given the limit of 5 figures for short report the authors can consider moving Figure 5 showing gene expression in presence of CX3CL1, as well as Key Resources Table to supplement or possibly merging Figure 4 with Figure 2.