Lack of the myotendinous junction marker col22a1 results in posture and locomotion disabilities in zebrafish.

The myotendinous junction (MTJ) is essential for the integrity of the musculoskeletal unit. Here, we show that gene ablation of the MTJ marker col22a1 in zebrafish results in MTJ dysfunction but with variable degrees of expression and distinct phenotypic classes. While most individuals reach adulthood with no overt muscle phenotype (class 1), a subset of the progeny displays severe movement impairment and die before metamorphosis (class 2). Yet all mutants display muscle weakness due to ineffective muscle force transmission that is ultimately detrimental for class-specific locomotion-related functions. Movement impairment at the critical stage of swimming postural learning causes class 2 larval death by compromising food intake. In class 1 adults, intensive exercise is required to uncover a decline in muscle performance, accompanied by higher energy demand and mitochondrial adaptation. This study underscores COL22A1 as a candidate gene for myopathies associated with dysfunctional force transmission and anticipates a phenotypically heterogeneous disease.

Pro-inflammatory immunity supports fibrosis advancement in epidermolysis bullosa: intervention with Ang-(1-7).

Recessive dystrophic epidermolysis bullosa (RDEB), a genetic skin blistering disease, is a paradigmatic condition of tissue fragility-driven multi-organ fibrosis. Here, longitudinal analyses of the tissue proteome through the course of naturally developing disease in RDEB mice revealed that increased pro-inflammatory immunity associates with fibrosis evolution. Mechanistically, this fibrosis is a consequence of altered extracellular matrix organization rather than that of increased abundance of major structural proteins. In a humanized system of disease progression, we targeted inflammatory cell fibroblast communication with Ang-(1-7)-an anti-inflammatory heptapeptide of the renin-angiotensin system, which reduced the fibrosis-evoking aptitude of RDEB cells. In vivo, systemic administration of Ang-(1-7) efficiently attenuated progression of multi-organ fibrosis and increased survival of RDEB mice. Collectively, our study shows that selective down-modulation of pro-inflammatory immunity may mitigate injury-induced fibrosis. Furthermore, together with published data, our data highlight molecular diversity among fibrotic conditions. Both findings have direct implications for the design of therapies addressing skin fragility and fibrosis.

Research in practice: Towards deciphering the role of epidermal proteases in recessive dystrophic epidermolysis bullosa progression.

Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable severe skin disease caused by loss of collagen VII, an extracellular protein that ensures skin cohesion. It manifests in skin blistering and unresolved cycles of wounding and healing that progressively lead to dermal stiffening and early development of aggressive cutaneous squamous cell carcinomas. Inflammation and subsequent tissue fibrosis highly contribute to RDEB pathogenicity and targeting them could provide new therapeutic options. Kallikreins (KLKs) are epidermal secreted proteases, which contribute to skin desquamation and inflammation. Kallikreins are involved in the pathogenesis of several inflammatory skin disorders, but interestingly also in the initiation and progression of different cancers. Our project aims at deciphering the role of KLKs in inflammation, fibrosis, and tumor development in RDEB.

QR-313, an Antisense Oligonucleotide, Shows Therapeutic Efficacy for Treatment of Dominant and Recessive Dystrophic Epidermolysis Bullosa: A Preclinical Study.

Dystrophic epidermolysis bullosa (DEB) is a blistering skin disease caused by mutations in the gene COL7A1 encoding collagen VII. DEB can be inherited as recessive DEB (RDEB) or dominant DEB (DDEB) and is associated with a high wound burden. Perpetual cycles of wounding and healing drive fibrosis in DDEB and RDEB, as well as the formation of a tumor-permissive microenvironment. Prolonging wound-free episodes by improving the quality of wound healing would therefore confer substantial benefit for individuals with DEB. The collagenous domain of collagen VII is encoded by 82 in-frame exons, which makes splice-modulation therapies attractive for DEB. Indeed, antisense oligonucleotide-based exon skipping has shown promise for RDEB. However, the suitability of antisense oligonucleotides for treatment of DDEB remains unexplored. Here, we developed QR-313, a clinically applicable, potent antisense oligonucleotide specifically targeting exon 73. We show the feasibility of topical delivery of QR-313 in a carbomer-composed gel for treatment of wounds to restore collagen VII abundance in human RDEB skin. Our data reveal that QR-313 also shows direct benefit for DDEB caused by exon 73 mutations. Thus, the same topically applied therapeutic could be used to improve the wound healing quality in RDEB and DDEB.

Kallikreins: Essential epidermal messengers for regulation of the skin microenvironment during homeostasis, repair and disease.

As the outermost layer of the skin, the epidermis is playing a major role in organism homeostasis providing the first barrier against external aggressions. Although considered as an extracellular matrix (ECM)-poor subtissue, the epidermal microenvironment is a key regulator of skin homeostasis and functionality. Among the proteins essential for upholding the epidermal microenvironment are the members of the kallikrein (KLK) family composed of 15 secreted serine proteases. Most of the members of these epithelial-specific proteins are present in skin and regulate skin desquamation and inflammation. However, although epidermal products, the consequences of KLK activities are not confined to the epidermis but widespread in the skin. In this review starting with the location and proteolytic activation cascade of KLKs, we present KLKs involvement in skin homeostasis, regeneration and pathology. KLKs have a large variety of substrates including ECM proteins, and evidence suggests that they are involved in the different steps of skin wound healing as discussed here. KLKs are also used as prognosis/diagnosis markers for many cancer types and we are focusing later on KLKs in cutaneous cancers, although their pathogenicity remains to be fully elucidated. Dysregulation of the KLK cascade is directly responsible for skin diseases with heavy inflammatory aspects, highlighting their involvement in skin immune homeostasis. Future studies will be needed to support the therapeutic potential of adjusting KLK activities for treatment of inflammatory skin diseases and wound healing pathologies.

A proinflammatory role of KLK6 protease in Netherton syndrome.

BACKGROUND: Netherton syndrome (NS) is a rare but severe type of ichthyosis characterized by atopy, allergies, and potentially lethal skin overdesquamation associated with highly elevated proteolytic activities in LEKTI-deficient epidermis. NS symptoms are recapitulated in Spink5-/- mouse where the gene encoding Lekti has been invalidated. Spink5-/- mice die within 5h from birth due to their severe skin barrier defect leading to dehydration. Spink5-/- mice also serve as a model for atopic dermatitis. The KLK6 protease is expressed by epidermal keratinocytes and shown in vitro to cleave desmosomal components. OBJECTIVE: To investigate in vivo whether KLK6 is implicated in epidermal overdesquamation and/or inflammation associated with NS. METHODS: The role of KLK6 was evaluated by generating Spink5-/-Klk6-/- double knockout mice. The phenotype was assessed by macroscopic observation, immunohistochemistry for differentiation markers, in situ zymography for proteolysis, and quantification of proinflammatory cytokines. RESULTS: Elimination of Klk6 in Spink5-/- remarkably suppresses the expression of Tslp, a major itching-inducing factor and driver of allergic reactions. Tnfα and the Th17 promoting cytokine Il-23 were also suppressed. Spink5-/-Klk6-/- mice display normalized keratinocyte differentiation, nevertheless, epidermal proteolytic activities and the associated overdesquamation were not ameliorated, and Spink5-/-Klk6-/- still died from a severe epidermal barrier defect as the Spink5-/-. CONCLUSIONS: Ablation of Klk6 largely suppresses epidermal inflammation but cannot rescue overdesquamation leading to the lethal NS phenotype. Nonetheless, our findings demonstrate for the first time that KLK6 is implicated in skin inflammation and may represent a novel druggable target for NS and other inflammatory conditions e.g. atopic dermatitis.

Fishing for collagen function: About development, regeneration and disease.

Collagens are the most abundant vertebrate extracellular matrix proteins. They form a superfamily of 28 members that show a remarkable diversity in molecular and supramolecular organization, tissue distribution and function and mutations in collagen genes result in a wide range of inherited connective tissue diseases. In the recent years, unexpected and very diverse regulatory and mechanical collagen functions have been reported. But the structural and functional landscape of the collagen superfamily is still far from being complete. Zebrafish has emerged over the last decades as a powerful model to interrogate gene function and there are numerous advantages of using zebrafish for collagen research, including recent advances in genome editing technologies and the characterization of the zebrafish matrisome. One can confidently predict that zebrafish will rapidly become a popular vertebrate model to investigate the role of collagens in development, disease and regeneration as discussed in this chapter.

Gene profile of zebrafish fin regeneration offers clues to kinetics, organization and biomechanics of basement membrane.

How some animals regenerate missing body parts is not well understood. Taking advantage of the zebrafish caudal fin model, we performed a global unbiased time-course transcriptomic analysis of fin regeneration. Biostatistics analyses identified extracellular matrix (ECM) as the most enriched gene sets. Basement membranes (BMs) are specialized ECM structures that provide tissues with structural cohesion and serve as a major extracellular signaling platform. While the embryonic formation of BM has been extensively investigated, its regeneration in adults remains poorly studied. We therefore focused on BM gene expression kinetics and showed that it recapitulates many aspects of development. As such, the re-expression of the embryonic col14a1a gene indicated that col14a1a is part of the regeneration-specific program. We showed that laminins and col14a1a genes display similar kinetics and that the corresponding proteins are spatially and temporally controlled during regeneration. Analysis of our CRISPR/Cas9-mediated col14a1a knockout fish showed that collagen XIV-A contributes to timely deposition of laminins. As changes in ECM organization can affect tissue mechanical properties, we analyzed the biomechanics of col14a1a-/- regenerative BM using atomic force microscopy (AFM). Our data revealed a thinner BM accompanied by a substantial increase of the stiffness when compared to controls. Further AFM 3D-reconstructions showed that BM is organized as a checkerboard made of alternation of soft and rigid regions that is compromised in mutants leading to a more compact structure. We conclude that collagen XIV-A transiently acts as a molecular spacer responsible for BM structure and biomechanics possibly by helping laminins integration within regenerative BM.

The in-silico zebrafish matrisome: A new tool to study extracellular matrix gene and protein functions.

Extracellular matrix (ECM) proteins are major components of most tissues and organs. In addition to their crucial role in tissue cohesion and biomechanics, they chiefly regulate various important biological processes during embryonic development, tissue homeostasis and repair. In essence, ECM proteins were defined as secreted proteins that localized in the extracellular space. The characterization of the human and mouse matrisomes provided the first definition of ECM actors by comprehensively listing ECM proteins and classified them into categories. Because zebrafish is becoming a popular model to study ECM biology, we sought to characterize the zebrafish matrisome using an in-silico gene-orthology-based approach. We report the identification of 1002 genes encoding the in-silico zebrafish matrisome. Using independent validations, we provide evidence for the robustness of the orthology-based approach. Moreover, we evaluated the orthology relationships between human and zebrafish genes at the whole-genome and matrisome levels and showed that the different categories of ECM genes are differentially subjected to evolutionary pressure. Last, we illustrate how the zebrafish matrisome list can be employed to annotate big data using the example of a previously published proteomic study of the skeletal ECM. The establishment of the zebrafish matrisome will undoubtedly facilitate the analysis of ECM components in "-omic" data sets.

Dual impact of live Staphylococcus aureus on the osteoclast lineage, leading to increased bone resorption.

BACKGROUND: Bone and joint infection, mainly caused by Staphylococcus aureus, is associated with significant morbidity and mortality, characterized by severe inflammation and progressive bone destruction. Studies mostly focused on the interaction between S. aureus and osteoblasts, the bone matrix-forming cells, while interactions between S. aureus and osteoclasts, the only cells known to be able to degrade bone, have been poorly explored. METHODS: We developed an in vitro infection model of primary murine osteoclasts to study the direct impact of live S. aureus on osteoclastogenesis and osteoclast resorption activity. RESULTS: Staphylococcal infection of bone marrow-derived osteoclast precursors induced their differentiation into activated macrophages that actively secreted proinflammatory cytokines. These cytokines enhanced the bone resorption capacity of uninfected mature osteoclasts and promoted osteoclastogenesis of the uninfected precursors at the site of infection. Moreover, infection of mature osteoclasts by live S. aureus directly enhanced their ability to resorb bone by promoting cellular fusion. CONCLUSIONS: Our results highlighted two complementary mechanisms involved in bone loss during bone and joint infection, suggesting that osteoclasts could be a pivotal target for limiting bone destruction.