1. Evaluation Summary:

    This study builds upon the previous original work of the authors by identifying a pathway that regulates collagen nanostructure and stiffness in lung fibrosis and demonstrating that this pathway it is independent of pathways regulating collagen synthesis. The analysis performed to validate the specificity of experimental findings and the demonstration that HIF activation is required for the increased tissue stiffness associated with fibrosis are elegant and convincing. With some additional clarifications and controls, this manuscript will be of broad interest to researchers interested in lung biology and especially to those focused on the pathogenesis and therapy of lung fibrosis.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    This paper demonstrates that the collagen crosslinking proteins PLOD2 and LOXL2 are significantly increased in IPF and investigates the upstream pathway involved in their regulation; this paper is based on previous work from this group showing that collagen crosslinking is key in regulating collagen structure and function, and tissue stiffness, in human lung fibrosis progression. The authors interrogate several known fibrotic pathways and find that DMOG, a hypoxia mimetic, is the most potent up-regulator of PLOD2/LOXL2 and that activation of the hypoxia-associated HIF pathway is required for expression of PLOD2/LOXL2, possibly acting synergistically with TGF-beta in the up-regulation of PLOD2. Although the authors consider the fact that LOX and LOXL1 are also increased, and justify their focus on LOXL2 by its correlation with PLOD2 expression, it may not be justified to completely ignore LOX and LOXL1. Critically, collagen deposition appears to be independently regulated from its crosslinking. They confirm that HIF pathway activation leads to the structural and mechanical changes they previously described. Interestingly, they show using published gene expression datasets that this pathway is up-regulated in 2 fibroblast subpopulations found in idiopathic pulmonary fibrosis and that pathway activation is independent of oxygen status. This adds to a recent publication reporting that HIF activation can occur in normoxic conditions and indeed, the authors demonstrate that oxidative stress can activate this pathway and thereby collagen crosslinking in IPF fibroblasts (with consistent findings in human IPF tissue). These data collectively confirm that collagen crosslinking is a key component of the changes in IPF and elucidates the novel pseudohypoxic pathway that regulates it. The data are overall solid and convincing.

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  3. Reviewer #2 (Public Review):

    Brereton et al. investigated the pathways potentially involved in the stiffening and accumulation of the ECM of lung fibrosis Following a study of the same group (Jones, 2018), they showed that Hypoxia-inducible factor (HIF) pathway was pathologically activated in sites of active fibrogenesis and responsible for " bone-type" pyridoline collagen cross-linking and altered collagen architecture. This alteration of the collagen architecture is responsible for the characteristic ECM stiffness of lung fibrosis responsible for ECM stiffness of lung fibrosis. In particular, they showed that LOXL2 and PLOD2 were responsible for collagen modification in the lung fibrosis foci.

    Weaknesses:
    1. the evidence that LOXL2 and PLOD2 are involved in collagen cross-linking is indirect. The authors need to show direct involvement of LOXL2 and PLOD2, e.g. using siRNA to revert the stiffness phenotype. They also should show co-localization of LOXL2 and PLOD2 in area of more collagen deposition (COL1A1 and COL 3A) since there is a synergistic effect between TGF beta and HIF pathways in IPF foci.

    2. The authors use primary lung fibroblasts for their experiments. They did not show any characterization of the donors for both healthy and IPF patients. The experiments of all the figures do not have always the right control. Since in IPF there is substantial genetic background, authors need to present data obtained from primary fibroblasts from at least 3 healthy as well as 3 IPF donors to support their hypothesis and conclusion. The selection criteria and characterization of the donors should be presented.

    3. mRNA is not always a prediction of protein expression levels. The mRNA data have to be confirmed with immunostaining/WB data.

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  4. Reviewer #3 (Public Review):

    This manuscript by Brereton et al. describes a specific role for Hypoxia-Induced Factor (HIF) in lung fibrosis. They discover that HIF is a critical factor that regulate post-translational modification of collagen, inducing abnormal 'bone-type' crosslinking, that cause the increased stiffness typical of fibrotic tissue. This mechanism is distinct from the known role of TGFb in increasing the collagen production in fibrosis.

    HIF pathway was found increased in human fibroblasts from patients with lung fibrosis, and this was associated with reduced expression of Factor Inhibiting HIF (FIH). Furthermore, oxidative stress, known to be increased in lung fibrosis, promoted HIF activation in normal fibroblasts, and was also found co-upregulated with HIF in mesenchymal cells derived from IPF lungs. These results show a link between oxidative stress and HIF pathway as a modulator of aberrant collagen crosslinking in lung fibrosis.

    The results of this manuscript are supported by well-designed experiments and clear results. The interpretation that HIF is acting in a pseudo hypoxic modality is not completely demonstrated by the data presented, but this do not reduce the importance of the finding that HIF is a critical modulator of crosslinking in lung fibrosis.

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