IL-6 signaling exacerbates hallmarks of chronic tendon disease by stimulating progenitor proliferation & migration to damage
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eLife assessment
This important study details an enrichment of the IL-6 signaling pathway in human tendinopathy, and applies transcriptional profiling to an advanced in vitro model to test IL-6 specific phenotypes in tendinopathy. Overall, the strength of evidence is solid yet incomplete, as transcriptomic measurements provide clarity, though functional studies including analysis of proliferation are needed to confirm these findings. This work will be of interest to stem cell biologists and immunologists.
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Abstract
Tendinopathies are debilitating diseases currently increasing in prevalence and associated costs. There is a need to deepen our understanding of the underlying cell signaling pathways to unlock effective treatments. In this work, we screen cell signaling pathways in human tendinopathies and find enriched IL-6/JAK/STAT signaling alongside signatures of cell populations typically activated by IL-6 in other tissues. To dissect the underlying causalities, we combine IL-6 knock-out mice with an explant-based assembloid model of tendon damage to successfully connect IL-6 signaling to fibroblast progenitor activation and recruitment. Vice versa, we show that these fibroblast progenitors promote the development of tendinopathy hallmarks in the damaged explant upon IL-6 activation. Finally, we present in vivo data confirming diminished migration of progenitors to acute Achilles tendon lesions in IL-6 knock-out mice. We conclude that IL-6 activates tendon tissues to initiate normal healing processes that can deteriorate into tendinopathy hallmarks.
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eLife assessment
This important study details an enrichment of the IL-6 signaling pathway in human tendinopathy, and applies transcriptional profiling to an advanced in vitro model to test IL-6 specific phenotypes in tendinopathy. Overall, the strength of evidence is solid yet incomplete, as transcriptomic measurements provide clarity, though functional studies including analysis of proliferation are needed to confirm these findings. This work will be of interest to stem cell biologists and immunologists.
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Reviewer #1 (Public Review):
This work by Stauber et al. is focused on understanding the signaling mechanisms that are associated with tendinopathy development, and by screening a panel of human tendinopathy samples, identified IL-6/JAK/STAT as a potential mediator of this pathology. Using an innovative explant model they delineated the requirement for IL-6 in the main body of the tendon to alter the dynamics of cells in the peritendinous synovial sheath space.
The use of a publicly available existing dataset is considered a strength since this dataset includes expression data from several different human tendons experiencing tendinopathy. This facilitates the identification of potentially conserved regulators of the tendinopathy phenotype.
The clear transcriptional shifts between WT and IL6-/- cores demonstrates the utility of the …
Reviewer #1 (Public Review):
This work by Stauber et al. is focused on understanding the signaling mechanisms that are associated with tendinopathy development, and by screening a panel of human tendinopathy samples, identified IL-6/JAK/STAT as a potential mediator of this pathology. Using an innovative explant model they delineated the requirement for IL-6 in the main body of the tendon to alter the dynamics of cells in the peritendinous synovial sheath space.
The use of a publicly available existing dataset is considered a strength since this dataset includes expression data from several different human tendons experiencing tendinopathy. This facilitates the identification of potentially conserved regulators of the tendinopathy phenotype.
The clear transcriptional shifts between WT and IL6-/- cores demonstrates the utility of the assembloid model, and supports the importance of IL6 in potentiating the cell response to this stimuli.
There are two main concerns with the manuscript in its current form:
First, the experimental approach does not directly assess proliferation, as such the conclusions regarding proliferation are not well supported. In the ex-vivo model, the use of cell counting approaches is somewhat acceptable since the system is constrained by the absence of potential influx of new cells. However, given the nearly unlimited supply of extrinsically derived cells in vivo (vs. the explant model), assessment of actual proliferation (e.g. Edu, BrdU, Ki67) is critical to support this conclusion.Second, the justification for the use of Scx-GFP+ cells as a progenitor population is not well supported. Indeed, in the discussion, Scx+ cells are treated as though they are uniformly a progenitor population, when the diversity of this population has been established by the cited studies, which do not suggest that these are progenitor populations. Additional definition/ delineation of these cells to identify the subset of these cells that may actually display other putative progenitor markers would support the conclusions. As it stands, the study currently provides important information on the impact of IL6 on Scx+ cells, but not tendon progenitors.
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Reviewer #2 (Public Review):
The authors of this study describe a goal of elucidating the signaling pathways that are upregulated in tendinopathy in order to target these pathways for effective treatments. Their goal is honorable, as tendinopathy is a common debilitating condition with limited treatments. The authors find that IL-6 signaling is upregulated in human tendinopathy samples with transcriptomic and GSEA analyses. The evidence of their initial findings are strong, providing a clinically-relevant phenotype that can be further studied using animal models.
Along these lines, the authors continue with an advanced in vitro system using the mouse tail tendon as the core with progenitors isolated from the Achilles tendon as the external sheath embedded in a hydrogel matrix. One question that comes to mind is whether the fibroblast …
Reviewer #2 (Public Review):
The authors of this study describe a goal of elucidating the signaling pathways that are upregulated in tendinopathy in order to target these pathways for effective treatments. Their goal is honorable, as tendinopathy is a common debilitating condition with limited treatments. The authors find that IL-6 signaling is upregulated in human tendinopathy samples with transcriptomic and GSEA analyses. The evidence of their initial findings are strong, providing a clinically-relevant phenotype that can be further studied using animal models.
Along these lines, the authors continue with an advanced in vitro system using the mouse tail tendon as the core with progenitors isolated from the Achilles tendon as the external sheath embedded in a hydrogel matrix. One question that comes to mind is whether the fibroblast progenitors in the extrinsic sheath of Achilles tendon is similar to those surrounding the tail tendon. The similarity of progenitors between different tendons is assumed with this model. I would consider this to be a minor issue, and would consider the in vitro system to be an additional strength of this study.
In order to address the IL-6 signaling pathway, the authors use core tendons from IL-6 knockout mice and progenitors from wild-type mice. The reasoning behind this approach was a little confusing... is IL-6 expressed solely in the tendon core compared to the extrinsic sheath? Furthermore, is a co-culture system for 7 days appropriate to model tendinopathy without the supplementation of exogenous inflammatory compounds? The transcriptomic differences in Figure 3 seem to be subtle, and may perhaps suggest that it could be a model that more closely resembles steady state compared to tendinopathy. If so, is IL-6 still relevant during steady state?
Nevertheless, the results presented in Figures 4 and 5 are impressive, demonstrating a link between IL-6 and fibroblast progenitor numbers and migration. Their experimental design in these figures show strong evidence, using Tocilizumab and recombinant IL-6 to rescue shown phenotypes. I would reduce the claims on proliferation, however, unless a proliferation-specific marker (e.g., Ki67, BrdU, EdU) is included in confocal analyses of Scx+ progenitors. The Achilles tendon injury model provides a nice in vivo confirmation of Scx-progenitor migration to the neotendon.
Given their goal to elucidate signaling pathways that could be targeted in the clinic, I think it would significantly strengthen the study if they could measure tendon healing in IL-6 knockouts or in wild-type mice treated with IL-6 inhibitors, since conventional ablation of IL-6 may lead to the elevation of compensatory IL-6 superfamily ligands that could activate STAT signaling. The authors claim that reducing IL-6 signaling decreases transcriptomic signatures of tendinopathy, but IL-6 may be necessary to promote normal healing of the tendon following injury. It is supposed that a lack of Scx+ progenitor migration would delay tendon healing.
Overall, the authors of this study elucidated IL-6 signaling in tendinopathy and provided a strong level of evidence to support their conclusions at the transcriptomic level. However, functional studies are needed to confirm these phenotypes and fully support their aims and conclusions. With these additional studies, this work has the potential to significantly influence treatments for those suffering from tendinopathy.
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