Dimethyl fumarate and 4-octyl itaconate are anticoagulants that suppress Tissue Factor in macrophages via inhibition of Type I Interferon

Excessive inflammation-associated coagulation is a feature of infectious diseases, occurring in such conditions as bacterial sepsis and COVID-19. It can lead to disseminated intravascular coagulation, one of the leading causes of mortality worldwide. Recently, type I interferon (IFN) signaling has been shown to be required for tissue factor (TF; gene name F3) release from macrophages, a critical initiator of coagulation, providing an important mechanistic link between innate immunity and coagulation. The mechanism of release involves type I IFN-induced caspase-11 which promotes macrophage pyroptosis. Here we find that F3 is a type I IFN-stimulated gene. Furthermore, F3 induction by lipopolysaccharide (LPS) is inhibited by the anti-inflammatory agents dimethyl fumarate (DMF) and 4-octyl itaconate (4-OI). Mechanistically, inhibition of F3 by DMF and 4-OI involves suppression of Ifnb1 expression. Additionally, they block type I IFN- and caspase-11-mediated macrophage pyroptosis, and subsequent TF release. Thereby, DMF and 4-OI inhibit TF-dependent thrombin generation. In vivo, DMF and 4-OI suppress TF-dependent thrombin generation, pulmonary thromboinflammation, and lethality induced by LPS, E. coli, and S. aureus, with 4-OI additionally attenuating inflammation-associated coagulation in a model of SARS-CoV-2 infection. Our results identify the clinically approved drug DMF and the pre-clinical tool compound 4-OI as anticoagulants that inhibit TF-mediated coagulopathy via inhibition of the macrophage type I IFN-TF axis.

This manuscript has been previously reviewed at another journal that is not operating a transparent peer review scheme. This document only contains reviewer comments and rebuttal letters for versions considered at Nature Communications.

Reviewer #2 (Remarks to the Author):
The authors provide compelling new data that addresses many of the concerns initially raised in my review. The new experiments bolster the model proposed and make a compelling story. I have no further issues.

Reviewer #3 (Remarks to the Author):
Dr Ryan and colleagues report in this paper on the impact of dimethyl fumarate (DMF) and an Itaconate derivate (4-OI) on the tissue factor dependent (TF) activation of the coagulation system. The main findings of this study are i) DMF and 4-OI inhibit TF-mediated coagulopathy via inhibition of the macrophage type I IFN-TF axis. The authors are to be congratulated for an extensive work that has been done for the revision of this manuscript. I enjoyed reading your report. The authors addressed very elegantly all of my comments.

Reviewer #4 (Remarks to the Author):
Ryan and colleagues describe how the type I interferon (IFN-I)-TF axis in macrophages can be modulated by DMF and 4-OI. They also prove that DMF and 4-OI inhibition of IFN-I also prevents caspase-11 induction. The authors use several mouse models of inflammation and infection and convincingly show that utilization of DMF and 4-OI is an effective intervention to decrease thromboinflammation.
The authors have been responsive to the original critiques and strongly improved their manuscript. There is only one point that in my opinion may need to be further amended or better discussed.
As also reported in previous reviews, it is hard to understand whether the anti-inflammatory activity of DMF and 4-OI is solely, or mostly, due to the inhibition of IFN-I (and its consequences on TF and caspase-11) or whether there is a general inhibition of the immune response due to the block of other inflammatory pathways.
Although the authors show that NRF2 is activated, this may be expected due to the fact that both DMF and 4OI activate NRF2. I would thus suggest to: i) if the authors have the data, show other pro-or anti-inflammatory cytokine levels for their in vitro and in vivo (as they already did in Sup Fig. 6a,b for the CoV2 infection) experiments; ii) if this is not possible in the short term, extensively discuss and highlight in their discussion that the anti-inflammatory effects in response to DMF and 4OI may also be due to other activities beside inhibition of the IFN-I-TF/Caspase-11 pathway.
Finally, Figure 3G reports Caspase-11KO mice, but the text specifies that an inhibitor has been utilized. The authors may want to consolidate this discrepancy.

Referee #2 -Remarks to the Author:
The authors provide compelling new data that addresses many of the concerns initially raised in my review. The new experiments bolster the model proposed and make a compelling story. I have no further issues.
Referee #3 -Remarks to the Author: Dr Ryan and colleagues report in this paper on the impact of dimethyl fumarate (DMF) and an Itaconate derivate (4-OI) on the tissue factor dependent (TF) activation of the coagulation system. The main findings of this study are i) DMF and 4-OI inhibit TF-mediated coagulopathy via inhibition of the macrophage type I IFN-TF axis. The authors are to be congratulated for an extensive work that has been done for the revision of this manuscript. I enjoyed reading your report. The authors addressed very elegantly all of my comments.
Referee #4 -Remarks to the Author: Ryan and colleagues describe how the type I interferon (IFN-I)-TF axis in macrophages can be modulated by DMF and 4-OI. They also prove that DMF and 4-OI inhibition of IFN-I also prevents caspase-11 induction. The authors use several mouse models of inflammation and infection and convincingly show that utilization of DMF and 4-OI is an effective intervention to decrease thromboinflammation.
The authors have been responsive to the original critiques and strongly improved their manuscript. There is only one point that in my opinion may need to be further amended or better discussed.
As also reported in previous reviews, it is hard to understand whether the anti-inflammatory activity of DMF and 4-OI is solely, or mostly, due to the inhibition of IFN-I (and its consequences on TF and caspase-11) or whether there is a general inhibition of the immune response due to the block of other inflammatory pathways.
Although the authors show that NRF2 is activated, this may be expected due to the fact that both DMF and 4OI activate NRF2. I would thus suggest to: i) if the authors have the data, show other pro-or antiinflammatory cytokine levels for their in vitro and in vivo (as they already did in Sup Fig. 6a,b for the CoV2 infection) experiments; ii) if this is not possible in the short term, extensively discuss and highlight in their discussion that the anti-inflammatory effects in response to DMF and 4OI may also be due to other activities beside inhibition of the IFN-I-TF/Caspase-11 pathway.
Finally, Figure 3G reports Caspase-11KO mice, but the text specifies that an inhibitor has been utilized. The authors may want to consolidate this discrepancy.
We wish to record our appreciation to the reviewers and express our gratitude for their time and suggestions which have enhanced our manuscript. In addition, we thank the editor for their guidance and contributions throughout this peer review process. Building on the feedback from the reviewers, we hope we have now addressed all of their comments, for which we provide a point-by-point response below.

Point-by-point author response to comments from Referee #2:
The authors provide compelling new data that addresses many of the concerns initially raised in my review. The new experiments bolster the model proposed and make a compelling story. I have no further issues.
We thank reviewer #2 for their helpful comments which we believe have contributed to a stronger and more streamlined manuscript. We are pleased that we have successfully addressed many of the previous comments and that the reviewer found our new data to be compelling and worthy of publication.

Point-by-point author response to comments from Referee #3:
Dr Ryan and colleagues report in this paper on the impact of dimethyl fumarate (DMF) and an Itaconate derivate (4-OI) on the tissue factor dependent (TF) activation of the coagulation system. The main findings of this study are i) DMF and 4-OI inhibit TF-mediated coagulopathy via inhibition of the macrophage type I IFN-TF axis. The authors are to be congratulated for an extensive work that has been done for the revision of this manuscript. I enjoyed reading your report. The authors addressed very elegantly all of my comments.
We are grateful to the reviewer and their guidance with clarifying the focus on thromboinflammation within our manuscript. We are glad that the reviewer appreciates our extensive revisions and we are particularly pleased that they found our revised manuscript both enjoyable and elegant.

Point-by-point author response to comments from Referee #4:
Ryan and colleagues describe how the type I interferon (IFN-I)-TF axis in macrophages can be modulated by DMF and 4-OI. They also prove that DMF and 4-OI inhibition of IFN-I also prevents caspase-11 induction. The authors use several mouse models of inflammation and infection and convincingly show that utilization of DMF and 4-OI is an effective intervention to decrease thromboinflammation. caspase-11) or whether there is a general inhibition of the immune response due to the block of other inflammatory pathways.
Although the authors show that NRF2 is activated, this may be expected due to the fact that both DMF and 4OI activate NRF2. I would thus suggest to: i) if the authors have the data, show other pro-or antiinflammatory cytokine levels for their in vitro and in vivo (as they already did in Sup Fig. 6a,b for the CoV2 infection) experiments; ii) if this is not possible in the short term, extensively discuss and highlight in their discussion that the anti-inflammatory effects in response to DMF and 4OI may also be due to other activities beside inhibition of the IFN-I-TF/Caspase-11 pathway.
We agree with the reviewer on this point and thank them for highlighting this. To acknowledge this point, we have amended the penultimate paragraph of our discussion to include the lines (new additions underlined): "Collectively, our work supports recent clinical trials testing anti-inflammatory compounds as potential inhibitors of inflammation-associated coagulation. There is an extensive crosstalk between inflammation and coagulation and therefore the broad reduction in inflammation exerted by DMF and 4-OI may contribute to their anticoagulant properties. However, our study identifies a new, targetable pathway (the type I IFN-TF axis) in thromboinflammation and our work supports redeploying the clinically approved DMF in clinical trials for inflammation-associated coagulopathies, in particular for autoimmune and thromboinflammatory diseases including systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS), which are characterized by excessive type I IFN production and are associated with significantly greater risk of developing thrombotic cardiovascular disease".
We hope with these additions that we have suitably addressed the reviewer's helpful comment.
Finally, Figure 3G reports Caspase-11KO mice, but the text specifies that an inhibitor has been utilized.