EGFR kinase activity is required for TLR4 signaling and the septic shock response

Transcription

1 Manuscript EMBOR EGFR kinase activity is required for TLR4 signaling and the septic shock response Saurabh Chattopadhyay, Manoj Veleeparambil, Darshana Poddar, Samar Abdulkhalek, Sudip K. Bandyopadhyay, Volker Fensterl and Ganes C. Sen Corresponding author: Ganes C. Sen, Cleveland Clinic Review timeline: Submission date: 08 March 2015 Editorial Decision: 01 April 2015 Revision received: 12 June 2015 Editorial Decision: 28 July 2015 Revision received: 10 August 2015 Accepted: 17 August 2015 Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.) Editor: Nonia Pariente 1st Editorial Decision 01 April 2015 Thank you for your submission to EMBO reports. We have now received the three enclosed reports on it. As you will see, although all the referees find the topic of interest and in principle suitable for us, they also consider the study is incomplete and insufficiently conclusive at this stage. Given that all referees provide constructive suggestions on how to make the work more conclusive and insightful, I would like to give you the opportunity to revise your manuscript. If the referee concerns can be adequately addressed, we would be happy to accept your manuscript for publication. However, please note that it is EMBO reports policy to undergo one round of revision only and thus, acceptance of your study will depend on the outcome of the next, final round of peerreview. Revised manuscripts must be submitted within three months of a request for unless a short extension has been discussed with the editor; they will otherwise be treated as new submissions. When preparing your revision, please note that you manuscript will be published in full article format. This means that all materials and methods must be included in the main text, and results and discussion sections kept separate (as you have them). European Molecular Biology Organization 1

2 I look forward to seeing a revised form of your manuscript when it is ready. In the meantime, please contact me if I can be of any assistance. REFEREE REPORTS: Referee #1: In the manuscript "EGFR kinase activity is required for TLR4 signaling and the septic shock response", the authors investigate whether EGFR is important for the transcriptional responses induced by LPS stimulation. The paper is well written and the authors demonstrate that the activity of EGFR is physiologically relevant in that EGFR inhibitors can protect mice against LPS-induced septic shock However, there are a number of problems with interpretation of the biochemical data presented, and alternative explanations for the observations made are not fully considered. Major Comments: 1. Using chemical inhibitors and shrna depletion, the authors show that the kinase activity of EGFR is required for the expression of a subset of LPS-dependent genes. The authors argue that Gefitinb, an EGFR inhibitor, specifically inhibits a subset of LPS-induced genes and does not affect MYD88-dependent genes like TNF. However, Figure 2A suggests that the kinetics of TNF expression are delayed in the presence of Gefitinib (Gf) (i.e, in the absence of Gf TNF expression peaks at 2 hrs while in the presence of Gf TNF peaks at 6 hrs). A more detailed analysis of the kinetics of LPS induced genes may reveal an additional phenotype. Furthermore, there is no discussion about how the expression of IL-1beta, a MYD88-dependent gene, is controlled by EGFR kinase activity. 2. In figure 3, the authors examine the GF-dependent inhibition in MYD88-/- cells. Given the wide body of literature arguing that TNF is a MYD88-dependent gene, I am surprised that LPS still induced a strong TNF response in the absence of MYD88. These responses should be examined side-by-side with matched wild-type cells. 3. Figures 4-6 are the most confusing figures. While the authors nicely show that EGFR is required for both AKT phosphorylation and IRF3-phosphorylation/ nuclear accumulation, the authors attempt to argue that the primary role of EGFR is to activate AKT and subsequently B-catenin. However, the role of EGFR in promoting IRF3 nuclear accumulation cannot be ignored. The data support that AKT activity is required for B-catenin phosphorylation/ nuclear accumulation, but that EGFR may have multiple roles in activating this signaling pathway. 4. The data presented suggests that signaling from the plasma membrane by MYD88 is intact whereas signaling from TRIF on endosomes in blocked. Since EGFR is a known to promote endocytosis, why have the authors not considered the possibility that their inhibitors somehow block LPS-induced TLR4 endocytosis? Ample literature exists to support this idea, as blocking TLR4 endocytosis blocks TRIF signaling, but leaves MYD88 signaling intact. The authors are encouraged to 1) determine if TLR4 endocytosis is influenced by EGFR, and 2) determine if EGFR endocytosis is influenced by LPS treatment. 5. Is EGFR activated in response to LPS? If so, what is the ligand? Minor Points: -Statistics should be performed on transcriptions European Molecular Biology Organization 2

3 Referee #2: This paper suggests a novel form of interaction between the TLR4 and EGFR signaling pathways. The authors provide data that the EGFR tyrosine kinase activity is necessary selectively for the activation of TRIF dependent transcription after stimulation with LPS. EGFR kinase inhibitors such as Gefitinib prevented LPS-induced Akt and IRF3 phosphorylation leading to reduced transcription of IRF-induced genes such as Ifit1 and IFNb. They propose a mechanism by which EGFR signaling is required for PI3K-dependent activation of b-catenin, which in turn is required as a co-activator for IRF-mediated transcription, a finding that they partly had already reported in a previous paper. The authors also provide in vivo relevance for the interaction between TLR4 and EGFR by demonstrating reduced LPS-induced sepsis in mice in the presence of Gefitinib. The findings are interesting and most of the experiments are conclusive. However, the mechanism how TLR 4 and EGFR signaling interact is only poorly clarified. Major points 1) Mechanism of LPS-mediated EGFR transactivation should be better analyzed. The rapid response of IRF phosphorylation and transcriptional induction of IFN target genes indicates that EGFR signaling is ready to be turned on in these cells. This might be explained by EGFR ligands present in the serum of the culture medium. The use of serum free culture conditions and EGFR ligands in combination with LPS could clarify some of these questions. Alternatively, EGFR transactivation could be a possible mechanism. Does LPS stimulation lead to EGFR phosphorylation? If yes, how? Are EGFR ligands or the proteases that cleave them induced by LPS treatment? Is there direct interaction and activation? Despite the fact that Gefitinib is used in a lot of experiments, the authors never show that EGFR is expressed in the cells employed (except for KD experiments), nor do they show inhibition of EGFR phosphorylation after Gefitinib treatment. Additionally the use of another, more specific EGFR inhibitor, like Cetuximab, that inhibits ligand binding, should also be considered to address some of these questions. 2) LPS-mediated TLR4 signaling can occur at the level of the cell membrane (via Myd88 and TIRAP) and at the level of the endosome (via TRIF and TRAM). Only the endosomal TLR4 signaling seems to be affected by the EGFR. Where are the two receptors localized in the studied cells before and after LPS stimulation? The authors might want to explain how and where the interaction/transactivation of TLR4 and EGFR occurs (at the level of membrane or endosome?). 3) For all the results showing RNA expression or cytokine production etc. (Fig. 1, 2, 3, 6 and Suppl Fig S1 and S2) there is no statistical analysis mentioned (no p values etc.) Are the differences observed statistically significant? Minor points 4) In Figure 3, the experiment performed with Tunicamycin provides only indirect evidence for the importance of the TRIF adaptor. The use of TRIF knockout macrophages would be preferable to confirm this finding. 5) The reduction of IRF3 in the nuclear fraction of Gf treated cells (Fig. 5B) does not look convincing and seems like a blotting problem. Referee #3: This MS is a fine extension of published previous work describing, that the TLR3 driven TRIF signalling pathway requires EGFR kinase activity. Because Lps sensing TLR4 activation is critical for sepsis induction, and because Lpps triggers via TLR4 the independently operating MyD88 and TRIF pathway,the authors are able to recapitulate and extend their published TLR3-TRIF-EGFR data in the TLR4-TRIF system.specifically they show that specific EGFR kinase inhibitors block the Tlr4 driven PI3/Akt pathway required for activation of the co-factor beta-catenin,that in turn is needed for phosphorilation of TRIF at Ser 552. The signal pathways adressed have been described/published in other systems. However, that TLR4 uses in Lps driven septic schock EGFR to activate PI3/AKT is novel, and important.the data given are fine, yet somehow incomplete. The authors are urged to adress the question whether ligand activated TLR4 recruits EGFR (Coprecipitation experiments)if so TLR4 might be a target of EGFR. European Molecular Biology Organization 3

4 1st Revision - authors' response 12 June 2015 Reviewers' Comments: Referee #1: In the manuscript "EGFR kinase activity is required for TLR4 signaling and the septic shock response", the authors investigate whether EGFR is important for the transcriptional responses induced by LPS stimulation. The paper is well written and the authors demonstrate that the activity of EGFR is physiologically relevant in that EGFR inhibitors can protect mice against LPS-induced septic shock However, there are a number of problems with interpretation of the biochemical data presented, and alternative explanations for the observations made are not fully considered. Major Comments: 1. Using chemical inhibitors and shrna depletion, the authors show that the kinase activity of EGFR is required for the expression of a subset of LPS-dependent genes. The authors argue that Gefitinb, an EGFR inhibitor, specifically inhibits a subset of LPS-induced genes and does not affect MYD88-dependent genes like TNF. However, Figure 2A suggests that the kinetics of TNF expression are delayed in the presence of Gefitinib (Gf) (i.e, in the absence of Gf TNF expression peaks at 2 hrs while in the presence of Gf TNF peaks at 6 hrs). A more detailed analysis of the kinetics of LPS induced genes may reveal an additional phenotype. Furthermore, there is no discussion about how the expression of IL-1beta, a MYD88-dependent gene, is controlled by EGFR kinase activity. As suggested, a more detailed kinetics of gene induction has now been done (new Figs 1A, 2A). We have now discussed why the induction of some NF-κB-driven genes might be EGFRdependent. 2. In figure 3, the authors examine the GF-dependent inhibition in MYD88-/- cells. Given the wide body of literature arguing that TNF is a MYD88-dependent gene, I am surprised that LPS still induced a strong TNF response in the absence of MYD88. These responses should be examined side-by-side with matched wild-type cells. The side-by-side comparison is now shown in the new Fig S4. It is known that the TRIF branch can also activate NF-κB (Ref#11, Yamamoto et al, Science 2003) 3. Figures 4-6 are the most confusing figures. While the authors nicely show that EGFR is required for both AKT phosphorylation and IRF3-phosphorylation/ nuclear accumulation, the authors attempt to argue that the primary role of EGFR is to activate AKT and subsequently B- catenin. However, the role of EGFR in promoting IRF3 nuclear accumulation cannot be ignored. The data support that AKT activity is required for B-catenin phosphorylation/ nuclear accumulation, but that EGFR may have multiple roles in activating this signaling pathway. We agree and now suggested multiple roles of EGFR in the Discussion. 4. The data presented suggests that signaling from the plasma membrane by MYD88 is intact whereas signaling from TRIF on endosomes in blocked. Since EGFR is a known to promote endocytosis, why have the authors not considered the possibility that their inhibitors somehow block LPS-induced TLR4 endocytosis? Ample literature exists to support this idea, as blocking TLR4 endocytosis blocks TRIF signaling, but leaves MYD88 signaling intact. The authors are encouraged to 1) determine if TLR4 endocytosis is influenced by EGFR, and 2) determine if EGFR endocytosis is influenced by LPS treatment. European Molecular Biology Organization 4

5 We considered this possibility but gefitinib did not inhibit TLR4 endocytosis (New Fig S5). The results with MyD88 KO cells (Fig 3) also suggest that TLR4 internalization was not affected by gefitinib because TNF was strongly induced in its presence. LPS had no effect on EGFR endocytosis. 5. Is EGFR activated in response to LPS? If so, what is the ligand? We could not detect any EGFR activation, as measured by its Tyr phosphorylation, in response to LPS treatment of cells. Minor Points: -Statistics should be performed on transcriptions Statistical analyses of the transcriptional data have now been added Referee #2: This paper suggests a novel form of interaction between the TLR4 and EGFR signaling pathways. The authors provide data that the EGFR tyrosine kinase activity is necessary selectively for the activation of TRIF dependent transcription after stimulation with LPS. EGFR kinase inhibitors such as Gefitinib prevented LPS-induced Akt and IRF3 phosphorylation leading to reduced transcription of IRF-induced genes such as Ifit1 and IFNb. They propose a mechanism by which EGFR signaling is required for PI3K-dependent activation of b-catenin, which in turn is required as a co-activator for IRF-mediated transcription, a finding that they partly had already reported in a previous paper. The authors also provide in vivo relevance for the interaction between TLR4 and EGFR by demonstrating reduced LPS-induced sepsis in mice in the presence of Gefitinib. The findings are interesting and most of the experiments are conclusive. However, the mechanism how TLR 4 and EGFR signaling interact is only poorly clarified. Major points 1) Mechanism of LPS-mediated EGFR transactivation should be better analyzed. The rapid response of IRF phosphorylation and transcriptional induction of IFN target genes indicates that EGFR signaling is ready to be turned on in these cells. This might be explained by EGFR ligands present in the serum of the culture medium. The use of serum free culture conditions and EGFR ligands in combination with LPS could clarify some of these questions. Alternatively, EGFR transactivation could be a possible mechanism. Does LPS stimulation lead to EGFR phosphorylation? If yes, how? Are EGFR ligands or the proteases that cleave them induced by LPS treatment? Is there direct interaction and activation? Despite the fact that Gefitinib is used in a lot of experiments, the authors never show that EGFR is expressed in the cells employed (except for KD experiments), nor do they show inhibition of EGFR phosphorylation after Gefitinib treatment. Additionally the use of another, more specific EGFR inhibitor, like Cetuximab, that inhibits ligand binding, should also be considered to address some of these questions. We did not observe any indication of LPS-mediated transactivation of EGFR, as measured by its phosphorylation. Experiments with serum free culture medium indicate that EGFR internalization promotes IFN gene induction by LPS (new Fig S7). The literature does not suggest any effect of LPS on EGFR ligand synthesis or processing. We have now shown, as suggested, the EGFR expression levels and the effect of gefitinib on EGFR phosphorylation (new Fig S1). As suggested, we have tested Cetuximab, which blocks EGF binding to cell surface EGFR only; it did not inhibit IFN induction by LPS suggesting that intracellular EGFR action promotes TLR4 signaling (new Fig S8). 2) LPS-mediated TLR4 signaling can occur at the level of the cell membrane (via Myd88 and TIRAP) and at the level of the endosome (via TRIF and TRAM). Only the endosomal TLR4 signaling seems to be affected by the EGFR. Where are the two receptors localized in the studied cells before European Molecular Biology Organization 5

6 and after LPS stimulation? The authors might want to explain how and where the interaction/transactivation of TLR4 and EGFR occurs (at the level of membrane or endosome?). We did not observe any physical interaction between EGFR and TLR4 (Fig 4A). LPS treatment triggered internalization of TLR4 and did not affect EGFR location; under our experimental conditions, EGFR was both on plasma and endosomal membranes. We believe that the functional interaction is between endosomal EGFR and endosomal TLR4. 3) For all the results showing RNA expression or cytokine production etc. (Fig. 1, 2, 3, 6 and Suppl Fig S1 and S2) there is no statistical analysis mentioned (no p values etc.) Are the differences observed statistically significant? Statistical analyses have now been included. Minor points 4) In Figure 3, the experiment performed with Tunicamycin provides only indirect evidence for the importance of the TRIF adaptor. The use of TRIF knockout macrophages would be preferable to confirm this finding. The TRIF KO experiment is already in the literature (Ref#25, Shenderov et al J Immunol 2014); we followed their protocol. 5) The reduction of IRF3 in the nuclear fraction of Gf treated cells (Fig. 5B) does not look convincing and seems like a blotting problem. The experiment has been repeated and the new results are shown in Fig 5B. Referee #3: This MS is a fine extension of published previous work describing, that the TLR3 driven TRIF signalling pathway requires EGFR kinase activity. Because Lps sensing TLR4 activation is critical for sepsis induction, and because Lpps triggers via TLR4 the independently operating MyD88 and TRIF pathway, the authors are able to recapitulate and extend their published TLR3-TRIF-EGFR data in the TLR4-TRIF system.specifically they show that specific EGFR kinase inhibitors block the Tlr4 driven PI3/Akt pathway required for activation of the co-factor beta-catenin,that in turn is needed for phosphorilation of TRIF at Ser 552. The signal pathways adressed have been described/published in other systems. However, that TLR4 uses in Lps driven septic schock EGFR to activate PI3/AKT is novel, and important. The data given are fine, yet somehow incomplete. The authors are urged to adress the question whether ligand activated TLR4 recruits EGFR (Coprecipitation experiments)if so TLR4 might be a target of EGFR. As suggested, we have performed the co-immunoprecipitation experiment and the results show that TLR4 and EGFR do not physically interact (Fig 4A). This suggests a more indirect functional interaction. 2nd Editorial Decision 28 July 2015 Thank you for the submission of your revised manuscript to our offices and please accept my apologies for the time we have needed to contact you with a decision on your study. Referees 2 and 3 (referee 1 was not available) have now returned their reports, which as you will see are not in agreement, which has prompted further discussion to make a decision on your study. Below you will find the reports of referees 2 and 3. While the latter is now supportive of publication, the former still has various issues that preclude him/her form supporting publication. On European Molecular Biology Organization 6

7 balance, we will not ask that you address all of them. However, a few do need to be addressed in a last round of minor revision before your study can be accepted for publication. Specifically, for a successful revision, we will require that: - you discuss the caveats of using HEK293 cells instead of macrophages/myeloid cells for some of the experiments - address point 7 from referee 2's report regarding supplementary figure 5, which needs to be strengthened (more cells shown, findings quantified, etc) - discuss previous literature supporting a role for EGFR in the induction of cytokines - provide a model summarizing the overall message of the study In addition, from an editorial point of view, some things also need to be taken care of: -please ensure that all relevant figures and supplementary figures have been generated according to proper statistical analysis procedures, and all figure legends include information on the number of independent experiments measured, the type of error bars used (SD, SE,...), statistical test applied to the data and exact P values considered significant. In figures 1-SF3, only information regarding the number of experiments is included. SF4-8 are missing all statistical information. We look forward to receiving a final version of your study as soon as it is ready, and always within the next four weeks. REFEREE REPORTS: Referee #2: This reviewer is not satisfied with the revision of the paper since many of the questions asked have not been adequately addressed (see point 1 of the previous revision). Little has been done towards a better mechanistic understanding of the pathways proposed in particular the elucidation of the mechanism how TLR 4 and EGFR signaling interact is still unclear. The observation that in serum starved medium LPS does not induce IFNb (Sup Fig 7A) points towards the need of EGFR ligands to induce EGFR and the suggested downstream pathways and target genes. However, this is still poorly examined. More mechanistic experiments could have been done around this finding. EGFR activation and the inhibitory effect of Gefitinib should have been shown in myeloid cells and not in HEK293 cells (Supl. Fig 1B). This is not what the reviewer asked for. This data are important to strengthen the evidence that the effects seen by EGFR inhibitors in myeloid cells are due to EGFR kinase inhibition. Moreover, this reviewer does not understand why the Co-IPs were performed in HEK293 cells which are not object of this study. These experiments should be performed in macrophages and/or myeloid cells used for the other studies in the paper. Signaling in HEK 293 cells might be completely different than in macrophages. None of the Western Blots in Fig. 4 includes EGFR and phospho-egfr analysis, which would also demonstrate to which extend EGFR phosphorylation is inhibited by Gefitinib is respect to Akt inhibition etc. Experiments with TRIF deficient BMDM should have been included similar to what performed in Fig. 3 with MyD88-/- cells. In Supl. Fig 4 the authors show how TNF production in Myd88 -/- cells compares to wild-type cells. European Molecular Biology Organization 7

8 This should be shown also for the other genes analyzed in Myd88-/- BMDM, which are shown in Fig. 3 such as IL1 and IFNs etc. In non-permeabilized cells it seems that there is more TLR4 signal on the cell surface after Gefitinib treatment? Is this a representative image (only one single cell is shown)? Moreover, the signal should be quantified before drawing conclusions. The same applies to permeabilized cells. The in vivo sepsis experiment is convincing. However, what about the expression of the other cytokines (analyzed in vitro) after in vivo LPS treatment in the presence of Gefitinib? Moreover, there have been several papers in the literature reporting EGFR requirement for the induction of cytokines. For example for IL-6 in macrophages there have been two papers published in J. Immunol in 2013 and Nat. Cell Biol which have not been mentioned by the authors. A model should be included that summarizes the major findings. Referee #3: This MS has been adaequately revised in view of the crticism raised by the reviewers.the revised version strongly favours the novel and unexpected interpretation, that EGFR kinase activity is required for TLR4 medited activation of the PI3k/Akt pathway, that in turn activates beta-catenin (an obligatory co-activator of IRF's). Since the authors provide compelling aruments/data that extracellular EGFR appears not to be involved, the authors conclude that a not yet understood "functional interaction of endodosomal EGFR and TLR4" comes into play.in other words, the data clearly show an essential role of EGFR kinase activity in TLR-4 driven type 1Interferon induction, yet the final answer of the mechanism is still lacking. Nevertheless, as it stands this well written MS provides novel and valid informations on TLR-4 mediated signalling. 2nd Revision - authors' response 10 August 2015 Thank you for editing our manuscript. We have revised it further following your instructions. The highlights of our revisions are as follows. The reasons and the caveats of using HEK293 cells have been discussed Fig S5 has been expanded and data quantified (new Fig S5B) Previous reports on EGFR and cytokine induction have been discussed and cited A working model has been provided and discussed (new Fig 7F) The missing statistical information has been provided Many original Western blot images have been deposited Uncompressed images have been provided for Figs 1E, 4D (new blot), 4E, 5D and 6C. Figs 5A and 5C have been replaced with less contrasted images I hope that the paper is now acceptable for publication. 3rd Editorial Decision 17 August 2015 I am very pleased to accept your manuscript for publication in the next available issue of EMBO reports. Thank you for your contribution to EMBO reports and congratulations on a successful publication. Please consider us again in the future for your most exciting work. European Molecular Biology Organization 8