The Epigenetic Reader Protein SP140 Regulates Dendritic Cell Activation, Maturation and Tolerogenic Potential

Round 1

Reviewer 1 Report

In this manuscript, the authors performed an extensive array of biological tests to identify the role of SP140 in dendritic cell biology. They found that reducing SP140 expression by GSK761 leads to a tolerogenic phenotype in the DCs. These DCs were able to stimulate regulatory responses in T cells. Overall the manuscript is well-written with a logical structure. There are a few points for discussion:

 

-          Why did the authors chose to add SP140 inhibitor during monocyte maturation to DCs, rather than during or after LPS stimulation?

-          Fig 3 & 4: Can you explain the increase in CD86 and HLA-DR in GSK761 cells (not stimulated with LPS)?

-          Figure 6: The T cells only control is not very relevant, so I would remove it. Did you have a positive control e.g. PHA? Also, did you measure the production of an anti-inflammatory cytokine such as IL-10 or TGFβ?

-          Line 188: “ … LPS or 24 hours (mDCs).” should be “… LPS for 24 hours (mDCs).”

-          Lines 376-382 are extremely similar to 328-335, please rewrite.

 

-          Lines 358-361: I agree it would be very interesting to see T cell proliferation. The transwell experiments mentioned are not explained, what do the authors want to do with this assay and why? If you don’t want to go into details then I would leave out the suggestion to do transwell experiments. Similar comment for lines 386-387, why do you want to know whether the effects are antigen-specific or not?

Author Response

I have uploaded a pint-by-point letter pdf

Author Response File: Author Response.pdf

Reviewer 2 Report

This is an interesting paper showing that the drug GSK761 is  a specific inhibitor of SP140, a key factor in stimulating theTh1 immune response, overexpressed  in autoimmune diseases. The authors investigate the role of GSK761 in the down regulation of the inflammatory-immune reaction “in vitro”, activated by DC. They found a clear inhibitory trend and suggest that SP140 could be an important therapeutic target in autoimmune diseases. In general terms this trend is shown by the data reported, but it is still “a trend” which must be confirmed and validated. The paper is well described and the methods are clearly reported. However some aspects deserves to be better explained before publication.

 

Comments:

Abstract: TNF and IL6 secretion is not “strongly reduced”. It is “reduced” of about 38-50% which is not strongly.

Fig.2B and lines 129-131 and 296-302

In this figure the DC-induced cytokine production is shown.  In some cases (TNF and  IL10) the decrement is significant at 24 hours, while in others at 4 hours (IL1, IL6, IFNg and IL8). Is it possible that the effect of GSK in some cases lasts only few hours? Please discuss.

For IL10 it seems that p< 0,001 may be unlikely, please control. Being a tolerogenic cytokine could this reveal a conflictual effect? Please discuss.

IL12p70 reduction is considered  (line 131), but is it significant? It seems NS

The decrease for  TNF and IL8  while significanti s lower than 50%. The trend is in line with authors’ hypothesis, but is the extent enough to ensure  a tolerogenic trend? Please discuss.

Fig. 3  frequency of CD 80, 83 decreases in iDC  but didn’t change in mDC for CD80; for CD86 it was found and increment in iDC  while it didn’t change in mDC. Frequency and expression show different patterns: for example for CD80:  while frequency didn’t change, expression is inhibited (about 50%) in the presence of GSK. Is this compatible with a tolerogenic trend? These data are very dfficult to interpret and authors should discuss them. (e.g. Is a 50% inhibition of expression in the same amount of positive cells enough to ensure a consistent inhibition of immune reaction?).

The same discussion should be reported for Tbet /FOXP3 frequency vs expression  in Fig.6

Line 245: Overall, these data suggest………The reviewer think that, considering the intriguing ratio between frequency and expression, and the extent of inhibition in cytokine production (hardly > 50%), this sentence is too speculative and must be less incisive (it may suggest a trend…..)

The authors themselves state that (Lines 365-366) GSK761 is not suitable to evaluate………as discussed in ref 10. In this  reference however no data are reported (Unfortunately, due to poor in vivo pharmokinetics (data not shown), GSK761 was not suitable to evaluate the efects of SP140 inhibition in in  vivo animal models of colitis…….) . The reviewer thinks, that explaining better (in detail) the poor pharmacokinetics of GSK may be useful. While the drug cannot be employed for “in vivo” study? Can it be used for MS model? (C1b presentation).

This is an interesting paper showing that the drug GSK761 is  a specific inhibitor of SP140, a key factor in stimulating theTh1 immune response, overexpressed  in autoimmune diseases. The authors investigate the role of GSK761 in the down regulation of the inflammatory-immune reaction “in vitro”, activated by DC. They found a clear inhibitory trend and suggest that SP140 could be an important therapeutic target in autoimmune diseases. In general terms this trend is shown by the data reported, but it is still “a trend” which must be confirmed and validated. The paper is well described and the methods are clearly reported. However some aspects deserves to be better explained.

 

Co0mments:

Abstract: TNF and IL6 secretion is not “strongly reduced”. It is “reduced” of about 38-50% which is not strongly.

Fig.2B and lines 129-131 and 296-302

In this figure the DC-induced cytokine production is shown.  In some cases (TNF and  IL10) the decrement is significant at 24 hours, while in others at 4 hours (IL1, IL6, IFNg and IL8). Is it possible that the effect of GSK in some cases lasts only few hours? Please discuss.

For IL10 it seems that p< 0,001 may be unlikely, please control. Being a tolerogenic cytokine could this reveal a conflictual effect? Please discuss.

IL12p70 reduction is considered  (line 131), but is it significant? It seems NS

The decrease for  TNF and IL8,  while significant is lower than 50%. The trend is in line with authors’ hypothesis, but is the extent enough to ensure  a tolerogenic trend? Please discuss.

Fig. 3  frequency of CD 80, 83 decreases in iDC  but didn’t change in mDC for CD80; for CD86 it was found and increment in iDC  while it didn’t change in mDC. Frequency and expression show different patterns: for example for CD80:  while frequency didn’t change, expression is inhibited (about 50%) in the presence of GSK. Is this compatible with a tolerogenic trend? These data are very dfficult to interpret and authors should discuss them. (e.g. Is a 50% inhibition of expression in the same amount of positive cells enough to ensure a consistent inhibition of immune reaction?).

The same discussion should be reported for Tbet /FOXP3 frequency vs expression  in Fig.6

Line 245: Overall, these data suggest………The reviewer think that, considering the intriguing ratio between frequency and expression, and the extent of inhibition in cytokine production (hardly > 50%), this sentence is too speculative and must be less incisive (it may suggest a trend…..)

The authors themselves state that (Lines 365-366) GSK761 is not suitable to evaluate………as discussed in ref 10. In this  reference however no data are reported (Unfortunately, due to poor in vivo pharmokinetics (data not shown), GSK761 was not suitable to evaluate the efects of SP140 inhibition in in  vivo animal models of colitis…….) . The reviewer thinks, that explaining better (in detail) the poor pharmacokinetics of GSK may be useful. While the drug cannot be employed for “in vivo” study? Can it be used for MS model? (C1b presentation).

Author Response

I have uploaded a pint-by-point letter pdf

Author Response File: Author Response.pdf