Metalloproteinases TACE and MMP-9 Differentially Regulate Death Factors on Adult and Neonatal Monocytes After Infection with Escherichia coli

Round  1

Reviewer 1 Report

The mansucript by Dreschers et al. Is well written and the conclusion is reached.

There are a few minor issues that should be addressed:

1. In the introductory lines 40-44 these sentences should be quoted with current literature.

2. The stated significances in the legends of the figures are all misleading: Significances should only be given with * to *** characters. **** and ***** signs are not relevant. In the figure legend, the ***** sign of significance (whatever that is) does not appear in the diagrams. The order of the significances in the figure legends is not correct. p<0.05, followed by 0.01 and then again followed by 0.05, makes no sense. This reviewer recommends reducing the significances to 0.05, 0.01, 0.001. Please indicate in the figure legend only the significances of the significances used in the diagrams.

Author Response

1. In the introductory lines 40-44 these sentences should be quoted with current literature.

 Response 1: We now added the requested quotation.

2. The stated significances in the legends of the figures are all misleading: Significances should only be given with * to *** characters. **** and ***** signs are not relevant. In the figure legend, the ***** sign of significance (whatever that is) does not appear in the diagrams. The order of the significances in the figure legends is not correct. p<0.05, followed by 0.01 and then again followed by 0.05, makes no sense. This reviewer recommends reducing the significances to 0.05, 0.01, 0.001. Please indicate in the figure legend only the significances of the significances used in the diagrams.

Response 2: We apologize for the inappropriate presentation of the statistical analysis. We now corrected our mistakes and followed the recommendations of the reviewer.

Reviewer 2 Report

In this manuscript, the authors studied how two metalloproteases TACE and MMP-9 regulate death factors on adult and neonatal monocytes post E. coli infection at different extent. The results are interesting and useful to help understand how the neonatal monocytes suppress excess inflammation to avoid cell death. I have several questions listed below.

1.    In Fig 1, they claimed that the TACE showed a significant increase in adult monocytes upon E. coli infection, whereas the MMP9 showed the upregulation in neonatal monocytes. I think if the authors can switch the order of Fig 1B and Fig 1C, it is more logical, since the Fig 1B tested the inhibitor of MMP9, but they did not show any MMP-9 data before that. For Figure 1D, the authors should carefully write the annotation of each gel slice. For example, there are two MMP-9 slices there, which one is which, regular SDS-PAGE gel staining or zymography. 

2.    In Fig 2, they stated that the inhibition of MMP-9 increased the expression of CD95 on the cell plasma membrane in adult and neonatal monocytes, whereas the inhibition of TACE did not show any effects. In contrast, the inhibition of MMP-9 showed no change of plasma membrane-localized TNFα, while inhibition of TACE increased the abundance of TNFα in both adult and neonatal cells. Interestingly, both inhibitions reduced the shedding of CD95 and TNFα. In Figure 2A and 2B, they tested the percentage of the cells expressing CD14+/memCD95 or CD14+/memTNF, but it is not clear if the expression of memCD95 and memTNF on the membrane is higher than non-treated. I suggest that the authors include such data in this Figure, the surface staining of both antibodies and quantified using flow cytometer will help gather such information to distinguish only the population becomes higher or the surface expression of both proteins becomes higher as well. 

3.    In Fig 3, the authors stated that the inhibition of MMP-9 increases PICD of CBMO. But it also happened in PBMO as well although the extent is much less than that in CBMO. The author can consider adding a ratio value of PICD increase after and before inhibition of MMP-9 in PBMO and CBMO, which can help the audience appreciate the difference in both cell lines. 

4.    In Fig 4, the authors tested that the addition of TNFα increased the population of memCD95-expressing monocytes. But I think it probably more appropriate if they can show that the level of memCD95 increases as well upon TNFα treatment.

5.    In Fig 5, they tested if the apoptosis of bystander monocytes is mediated in a cell-contact manner or not. This is very interesting. Using the transwell chamber assay, they measured the apoptosis rates in E. coli infected monocytes and non-contact cells. The results showed that the contact with E. coli can efficiently increase the apoptosis rate in monocytes. I suggest that the author may also consider testing the CD14+/memCD95 or CD14+/memTNF cell population in the upper chamber containing E. coli infected monocytes and the monocytes in the lower chamber which don’t directly interact with E. coli, but can receive the secreted TNF and CD95. Thus, we will have a better idea whether the monocytes having direct contact with E. coli show higher CD14+ than non-infected monocytes. 

Author Response

1.    In Fig 1, they claimed that the TACE showed a significant increase in adult monocytes upon E. coli infection, whereas the MMP9 showed the upregulation in neonatal monocytes. I think if the authors can switch the order of Fig 1B and Fig 1C, it is more logical, since the Fig 1B tested the inhibitor of MMP9, but they did not show any MMP-9 data before that. For Figure 1D, the authors should carefully write the annotation of each gel slice. For example, there are two MMP-9 slices there, which one is which, regular SDS-PAGE gel staining or zymography. 

Response 1: We thank the reviewer for this comment; we now restructured the figure according to the given recommendations. In panel D,  we added more precise annotations to the components presenting zymography and SDS PAGE control. We showed two different concentrations of protein lysates which are now specified next to  the panel.

2.    In Fig 2, they stated that the inhibition of MMP-9 increased the expression of CD95 on the cell plasma membrane in adult and neonatal monocytes, whereas the inhibition of TACE did not show any effects. In contrast, the inhibition of MMP-9 showed no change of plasma membrane-localized TNFα, while inhibition of TACE increased the abundance of TNFα in both adult and neonatal cells. Interestingly, both inhibitions reduced the shedding of CD95 and TNFα. In Figure 2A and 2B, they tested the percentage of the cells expressing CD14+/memCD95 or CD14+/memTNF, but it is not clear if the expression of memCD95 and memTNF on the membrane is higher than non-treated. I suggest that the authors include such data in this Figure, the surface staining of both antibodies and quantified using flow cytometer will help gather such information to distinguish only the population becomes higher or the surface expression of both proteins becomes higher as well.

Response 2: To address this question, we provided supplemental data (Supplemental Figure 3) on the surface expression of memCD95 and memTNF. PBMO showed reduced levels of memCD95 after treatment with both inhibitors and an increase of memCD95 after E.coli infection. CBMO showed no difference regarding memCD95 levels. The memTNF levels increased significantly after E.coli infection in PBMO and CBMO (Supplemental Figure 3).

We added this information to the result section (page 5, lines 137-140). Together with our already provided information we show, that only the population of PBMO and CBMO expressing memTNF and memCD95 is changing. To the Discussion Section, we added: We speculate that this regulation may be  phagocytosis dependent (see Fig. 5B), since the percentage of memCD95 appears higher on GFP+ phagocytosing monocytes (Fig. 5B) than on non-phagocytosing (GFP-) monocytes. This observation was one of the reasons for monitoring the shedding of CD95 and TNF via ELISA (Fig. 2C, D). We refer to this finding in the result section (page 11 lines 262-266). 

3.    In Fig 3, the authors stated that the inhibition of MMP-9 increases PICD of CBMO. But it also happened in PBMO as well although the extent is much less than that in CBMO. The author can consider adding a ratio value of PICD increase after and before inhibition of MMP-9 in PBMO and CBMO, which can help the audience appreciate the difference in both cell lines.

Response 3: We now have modified Figure 3 by additionally presenting a ratio of PICD values of the indicated groups providing evidence, that even after application of CHX the PICD in CBMO is lower compared to PBMO. We corrected the text in the result section (page 6 lines 176-177). Thank you for this valuable suggestion.

4.    In Fig 4, the authors tested that the addition of TNFα increased the population of memCD95-expressing monocytes. But I think it probably more appropriate if they can show that the level of memCD95 increases as well upon TNFα treatment.

Response 4: As requested, we added the mean fluorescence data to show that the level of memCD95 increases in the given experiments (new Figure 4B). Here, the presentation of the MFI values underline the specific and significant action of etanercept. This is now included in the manuscripts (page 7 lines197-200). Thank you again for this valuable suggestion.

5.    In Fig 5, they tested if the apoptosis of bystander monocytes is mediated in a cell-contact manner or not. This is very interesting. Using the transwell chamber assay, they measured the apoptosis rates in E. coli infected monocytes and non-contact cells. The results showed that the contact with E. coli can efficiently increase the apoptosis rate in monocytes. I suggest that the author may also consider testing the CD14+/memCD95 or CD14+/memTNF cell population in the upper chamber containing E. coli infected monocytes and the monocytes in the lower chamber which don’t directly interact with E. coli, but can receive the secreted TNF and CD95. Thus, we will have a better idea whether the monocytes having direct contact with E. coli show higher CD14+ than non-infected monocytes. 

Response 5: We now present the percentage of memCD95- and memTNF-positive monocytes and the mean concentration of memCD95 and memTNF on PBMO and CBMO from the trans-chamber (Fig. 5D). The percentage of memCD95-, memTNF-positive PBMO as well as the density of memTNF on PBMO was higher than on CBMO. In contrast, with less memCD95-positive CBMO, their mean density was higher than on PBMO (Fig. 5D). This is now included in the manuscript (page 9 lines 239-241) and discussed (page 11 lines 262-266).