Hypertrophic scars are fibroproliferative wound healing defects affecting up to 70% of patients with severe burns. Characterized by extracellular matrix (ECM) accumulation in the dermis, hypertrophic scars are often associated with an excessive production and activation of transforming growth factor (TGF)-Beta, a major pro-fibrotic cytokine. Granzyme B (GzmB) is a serine protease that is elevated in burn wounds and may contribute to scarring and fibrosis through the degradation of specific ECM proteins. GzmB notably cleaves Decorin, leading to the impairment of collagen fibrillogenesis and the subsequent release/activation of TGF-Beta. Furthermore, GzmB topical inhibition reduced scarring and decorin loss in a mouse model of burn wound healing. The present study aims at delineating the role of GzmB in hypertrophic scarring through the cleavage of the ECM, with a specific focus on TGF-Beta activation. Using skin sections from healthy controls (n=6) and patients with hypertrophic scars (n=10), we investigated GzmB ability to regulate TGF-Beta activation through the cleavage of extracellular matrix proteins. Immunohistochemical staining and co-immunofluorescence analyses showed an accumulation of GzmB-positive cells, especially mast cells, in the dermis of hypertrophic scar (n=10) patients compared to healthy controls (n=6). Staining for substance P, a neuropeptide involved in mast cell degranulation, suggests that GzmB is released extracellularly by mast cells. Compared to healthy skin, we confirmed the reduction of decorin and observed a loss of the ECM protein LTBP1 (Latent TGF-Beta Binding Protein-1) at the dermal-epidermal junction of hypertrophic scars. Using LTBP1 producing cells, its cleavage was confirmed by GzmB in vitro. Furthermore, increased activation of the TGF-beta/Smad signaling pathway was observed in cells treated with LTBP1 pre-digested with GzmB. This observation, abolished by a Pan-TGF-Beta inhibitor, indicates that GzmB promotes LTBP1 dependent TGF-Beta activation. GzmB may contribute to hypertrophic scar formation through multiple mechanisms involving ECM cleavage and TGF-Beta activation. This study will provide key rationale to pursue GzmB as a novel therapeutic target for the treatment of hypertrophic scars. This work was funded in part by grants-in-Aid from the Canadian Institutes for Health Research, National Sciences and Engineering Research Council of Canada, the British Columbia Professional Firefighters’ Burn Fund, and the International Brotherhood of Electrical Workers (IBEW) Local 258. One of the author is the recipient of the Arthritis Society Canada Training Postdoctoral Fellowship.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers due to late diagnosis and poor therapeutic efficiency. Throughout PDAC progression, a dense extracellular matrix (ECM) is deposited around neoplastic cells and accompanies tumor development and aggressiveness. This significant stroma, both in terms of quantity and through its impact on tumor cells, is now considered as a target for innovative therapies to improve patient survival. Among ECM proteins, Tenascins (TNs) are a family of four glycoproteins (TNC, TNW, TNR and TNX) sharing a common modular structure, but exhibiting different expression patterns and functions depending on the physiological and physio-pathological contexts. In PDAC, TNC is up-regulated and is considered as a pro-tumorigenic actor, whereas TNX role remains to be elucidated. Herein, we demonstrated that unlike TNC, TNX is drastically decreased in PDAC, and that this loss is correlated with reduced patient survival. Dysregulation of the TNX/TNC balance is attributable to Transforming Growth Factor-beta (TGF-β) upregulation during pancreatic carcinogenesis. Interestingly, we found that TNX is first highly deposited in low grade lesions before being clearly decreased in later stages suggesting an elaborate stromal remodelling during PDAC development. Finally, low TNX and high TNC deposition around precursor lesions are correlated with increased preneoplastic cell proliferation. Altogether, our results (i) demonstrate the importance of Tenascin ratios during pancreatic carcinogenesis, (ii) suggest an anti-proliferative role for TNX unlike its pro-tumoral counterpart, TNC and (iii) underscore TNX and TNC as valuable targets for the development of new drugs for pancreatic cancer and/or solid tumor treatment. ### Competing Interest Statement The authors have declared no competing interest.

Keloid scars (KS) and hypertrophic scars (HS) are fibroproliferative wound healing defects characterized by excessive accumulation of extracellular matrix (ECM) in the dermis of affected individuals. Although transforming growth factor (TGF)-β is known to be involved in the formation of KS and HS, the molecular mechanisms responsible for its activation remain unclear. In this study we investigated Granzyme B (GzmB), a serine protease with established roles in fibrosis and scarring through the cleavage of ECM proteins, as a potential new mediator of TGF-β activation in KS and HS. Increased GzmB-positive mast cells were identified in the dermis of KS and HS but not healthy skin controls. Elevated levels of substance P, a neuropeptide involved in mast cell degranulation, suggest that GzmB is released extracellularly, as confirmed by the significant reduction of the established extracellular GzmB substrate decorin in KS and HS. Similarly, presence of latent TGF-β binding protein 1 (LTBP1), a protein involved in the extracellular tethering of latent TGF-β, was disrupted proximal to the dermal-epidermal junction (DEJ) of GzmBhigh KS and HS lesions. Using LTBP1-enriched medium as well as purified LTBP1, its cleavage by GzmB was confirmed in vitro. Increased TGF-β/Smad signaling pathway was observed in keratinocytes treated with GzmB-digested LTBP1 and was abolished by the addition of a pan-TGF-β inhibitor, suggesting that GzmB cleavage of LTBP1 contributes to TGF-β activation. In dermal fibroblasts, GzmB also cleaved cell-derived LTBP1 and induced TGF-β activation through the cleavage of one or more unidentified fibroblast-secreted proteins. Altogether, the present results suggest that GzmB contributes to KS and HS through ECM remodeling and TGF-β activation.

Age-related macular degeneration (AMD) is a common retinal neurodegenerative disease among the elderly. Neovascular AMD (nAMD), a leading cause of AMD-related blindness, involves choroidal neovascularization (CNV), which can be suppressed by anti-angiogenic treatments. However, current CNV treatments do not work in all nAMD patients. Here we investigate a novel target for AMD. Granzyme B (GzmB) is a serine protease that promotes aging, chronic inflammation and vascular permeability through the degradation of the extracellular matrix (ECM) and tight junctions. Extracellular GzmB is increased in retina pigment epithelium (RPE) and mast cells in the choroid of the healthy aging outer retina. It is further increased in donor eyes exhibiting features of nAMD and CNV. Here, we show in RPE-choroidal explant cultures that exogenous GzmB degrades the RPE-choroid ECM, promotes retinal/choroidal inflammation and angiogenesis while diminishing anti-angiogenic factor, thrombospondin-1 (TSP-1). The pharmacological inhibition of either GzmB or mast-cell degranulation significantly reduces choroidal angiogenesis. In line with our in vitro data, GzmB-deficiency reduces the extent of laser-induced CNV lesions and the age-related deterioration of electroretinogram (ERG) responses in mice. These findings suggest that targeting GzmB, a serine protease with no known endogenous inhibitors, may be a potential novel therapeutic approach to suppress CNV in nAMD.

Psoriasis is an inflammatory disease with systemic manifestations that most commonly presents as itchy, erythematous, scaly plaques on extensor surfaces. Activation of the IL-23/IL-17 pro-inflammatory signaling pathway is a hallmark of psoriasis and its inhibition is key to clinical management. Granzyme K (GzmK) is an immune cell-secreted serine protease elevated in inflammatory and proliferative skin conditions. In the present study, human psoriasis lesions exhibited elevated GzmK levels compared to non-lesional psoriasis and healthy control skin. In an established murine model of imiquimod (IMQ)-induced psoriasis, genetic loss of GzmK significantly reduced disease severity, as determined by delayed plaque formation, decreased erythema and desquamation, reduced epidermal thickness, and inflammatory infiltrate. Molecular characterization in vitro revealed that GzmK contributed to macrophage secretion of IL-23 as well as PAR-1-dependent keratinocyte proliferation. These findings demonstrate that GzmK enhances IL-23-driven inflammation as well as keratinocyte proliferation to exacerbate psoriasis severity.

Rheumatoid arthritis (RA) is a common autoimmune disorder characterized by exacerbated joint inflammation. Despite the well-documented accumulation of the serine protease granzyme B (GzmB) in RA patient biospecimens, little is understood pertaining to its role in pathobiology. In the present study, tenascin-C (TNC) - a large, pro-inflammatory extracellular matrix glycoprotein - was identified as a substrate for GzmB in RA. GzmB cleaves TNC to generate 3 fragments in vitro: a 130 kDa fragment that remains anchored to the matrix and 2 solubilized fragments of 70 and 30 kDa. Mass spectrometry results suggested that the 30 kDa fragment contained the pro-inflammatory TNC C-terminal fibrinogen-like domain. In the synovial fluids of patients with RA, soluble levels of GzmB and TNC were significantly elevated compared with healthy controls. Further, immunoblotting revealed soluble 70 and 30 kDa TNC fragments in the synovial fluids of patients with RA, matching TNC fragment sizes generated by GzmB cleavage in vitro. Granzyme K (GzmK), another serine protease of the granzyme family, also cleaves TNC in vitro; however, the molecular weights of GzmK-generated TNC fragments did not correspond to TNC fragment sizes detected in patients. Our data support that GzmB, but not GzmK, contributes to RA through the cleavage of TNC.

Granzymes (granule-secreted enzymes) are a family of serine proteases that have been viewed as redundant cytotoxic enzymes since their discovery more than 30 years ago. Predominantly produced by cytotoxic lymphocytes and natural killer cells, granzymes are delivered into the cytoplasm of target cells through immunological synapses in cooperation with the pore-forming protein perforin. After internalization, granzymes can initiate cell death through the cleavage of intracellular substrates. However, evidence now also demonstrates the existence of non-cytotoxic, pro-inflammatory, intracellular and extracellular functions that are granzyme specific. Under pathological conditions, granzymes can be produced and secreted extracellularly by immune cells as well as by non-immune cells. Depending on the granzyme, accumulation in the extracellular milieu might contribute to inflammation, tissue injury, impaired wound healing, barrier dysfunction, osteoclastogenesis and/or autoantigen generation. Granzyme serine proteases are known for their perforin-dependent cytotoxic activities, but evidence also indicates that they have a range of non-cytotoxic, pro-inflammatory intracellular and extracellular functions. In this Review, the authors discuss granzyme biology with an emphasis on its involvement in rheumatic disease pathology. Granzymes are serine proteases with both cytotoxic and non-cytotoxic functions; phenotypic and mechanistic characterization of granzymes in rheumatic diseases is needed to delineate their specific roles.The five human granzymes have unique substrate specificities and functional roles, as determined by granzyme-specific cleavage preferences, location of accumulation (intracellular or extracellular) and exposure to substrates in tissues.Extracellular granzyme activity can contribute to tissue injury, inflammation, autoimmunity, epithelial and endothelial barrier dysfunction, bullae formation, impaired wound healing, and degenerative or pathological aging.In addition to cytoplasmic proteins involved in apoptosis, granzyme B substrates include extracellular matrix proteins, hemidesmosomal or desmosomal proteins, pro-inflammatory cytokines, cell surface receptors and autoantigens.Granzyme A and granzyme B are elevated in synovial fluid, tissues and plasma of people with rheumatoid arthritis; in an arthritis model, -/- mice have lower disease severity than that of wild-type mice.CD8+ T cells expressing granzyme B and granzyme K are enriched in the peripheral blood and inflamed tissues of people with rheumatoid arthritis, systemic lupus erythematosus or Sj & ouml;gren syndrome.

An in-depth understanding of the mechanical properties of the dermis is indispensable to improve wound healing or slow-down skin ageing. Despite crucial research issues for dermatological and cosmetic industries, very little is known about the mechanical behaviour of the dermis at nanoscale level. This knowledge is relevant not only to human skin but also to mouse skin since this animal model is widely used in basic and preclinical studies for skin biology and health. Here, we describe an original protocol that we developed to specifically measure the mechanical properties of mouse dermis using atomic force microscopy-based nano-indentation approach. Using horizontal cryosections (i.e. parallel to the skin surface) performed at different depths through the dermis of dorsal skin, our protocol allowed us to detect nanoscale mechanical changes between female and male dermis samples. We found that the dermis was softer (i) in females than in males and (ii) with depth within the dermis of male mice. We also quantified compositional differences between female and male skin dermis and found that increased extracellular matrix gene expression and type V collagen staining were associated with increased dermal stiffness in male mice, compared with females. Our results demonstrating a sexual dimorphism in the nanomechanical properties and molecular composition of mouse dermis, open the way to better consider sex-related cutaneous differences to understand skin disease and to stimulate the development of female versus male-specific products with more appropriate dermatological treatments and cosmetic interventions.

INTRODUCTION:Granzyme B is a serine protease extensively studied for its implication in cytotoxic lymphocyte-mediated apoptosis. In recent years, the paradigm that the role of granzyme B is restricted to immune cell-mediated killing has been challenged as extracellular roles for the protease have emerged. While mostly absent from healthy tissues, granzyme B levels are elevated in several autoimmune and/or chronic inflammatory conditions. In the skin, its accumulation significantly impairs proper wound healing.AREAS COVERED:After an overview of the current knowledge on granzyme B, a description of newly identified functions will be presented, focussing on granzyme B ability to promote cell-cell and dermal-epidermal junction disruption, extracellular matrix degradation, vascular permeabilization, and epithelial barrier dysfunction. Progress in granzyme B inhibition, as well as the use of granzyme B inhibitors for the treatment of tissue damage, will be discussed.EXPERT OPINION:The absence of endogenous extracellular inhibitors renders extracellular granzyme B accumulation deleterious for the proper healing of chronic wounds due to sustained proteolytic activity. Consequently, specific granzyme B inhibitors have been developed as new therapeutic approaches. Beyond applications in wound healing, other autoimmune and/or chronic inflammatory conditions related to exacerbated granzyme B activity may also benefit from the development of these inhibitors.

Pancreatic cancer is the seventh leading cause of cancer-related deaths worldwide and is predicted to become second in 2030 in industrialized countries if no therapeutic progress is made. Among the different types of pancreatic cancers, Pancreatic Ductal Adenocarcinoma (PDAC) is by far the most represented one with an occurrence of more than 90%. This specific cancer is a devastating malignancy with an extremely poor prognosis, as shown by the 5-years survival rate of 2-9%, ranking firmly last amongst all cancer sites in terms of prognostic outcomes for patients. Pancreatic tumors progress with few specific symptoms and are thus at an advanced stage at diagnosis in most patients. This malignancy is characterized by an extremely dense stroma deposition around lesions, accompanied by tissue hypovascularization and a profound immune suppression. Altogether, these combined features make access to cancer cells almost impossible for conventional chemotherapeutics and new immunotherapeutic agents, thus contributing to the fatal outcomes of the disease. Initially ignored, the Tumor MicroEnvironment (TME) is now the subject of intensive research related to PDAC treatment and could contain new therapeutic targets. In this review, we will summarize the current state of knowledge in the field by focusing on TME composition to understand how this specific compartment could influence tumor progression and resistance to therapies. Attention will be paid to Tenascin-C, a matrix glycoprotein commonly upregulated during cancer that participates to PDAC progression and thus contributes to poor prognosis.

Transforming growth factor-β (TGF-β) isoforms are secreted as inactive complexes formed through non-covalent interactions between bioactive TGF-β entities and their N-terminal pro-domains called latency-associated peptides (LAP). Extracellular activation of latent TGF-β within this complex is a crucial step in the regulation of TGF-β activity for tissue homeostasis and immune cell function. We previously showed that the matrix glycoprotein Tenascin-X (TN-X) interacted with the small latent TGF-β complex and triggered the activation of the latent cytokine into a bioactive TGF-β. This activation most likely occurs through a conformational change within the latent TGF-β complex and requires the C-terminal fibrinogen-like (FBG) domain of the glycoprotein. As the FBG-like domain is highly conserved among the Tenascin family members, we hypothesized that Tenascin-C (TN-C), Tenascin-R (TN-R) and Tenascin-W (TN-W) might share with TN-X the ability to regulate TGF-β bioavailability through their C-terminal domain. Here, we demonstrate that purified recombinant full-length Tenascins associate with the small latent TGF-β complex through their FBG-like domains. This association promotes activation of the latent cytokine and subsequent TGF-β cell responses in mammary epithelial cells, such as cytostasis and epithelial-to-mesenchymal transition (EMT). Considering the pleiotropic role of TGF-β in numerous physiological and pathological contexts, our data indicate a novel common function for the Tenascin family in the regulation of tissue homeostasis under healthy and pathological conditions.

BMP-1–mediated cleavage of TSP-1 reduces cell adhesion and promotes TGF-β signaling.

Les Ténascines sont des glycoprotéines complexes de la matrice extracellulaire regroupant quatre membres : la Ténascine-C (TN-C), la Ténascine-R (TN-R), laTénascine-W (TN-W) et la Ténascine-X (TN-X). Ces protéines ont des profils d’expression particulier chez l’adulte et sont souvent dérégulées au cours du développement tumoral. Il a été démontré que la TN-X, en plus d’avoir un rôle architectural au sein de la matrice extracellulaire, pouvait également réguler la signalisation cellulaire. En effet, la TN-X a la capacité d’activer le Transforming Growth Factor (TGF)-Beta latent, une cytokine secrétée par les cellules sous une forme inactive, grâce à son domaine C-terminal globulaire ressemblant au fibrinogène(FBG). Ce domaine FBG est hautement conservé entre les différents membres de la famille des Ténascines. Par conséquent, nous avons démontré que la TN-C, la TN-Rainsi que la TN-W pouvait également activer le TGF-Beta latent par l’intermédiaire deleur domaine FBG in vitro. Une telle activation permet la présentation du TGF-Betamature à la surface des cellules, aboutissant à l’activation d’une voie de signalisationcellulaire TGF-Beta/Smad fonctionnelle. Nous avons également démontré que lesdomaines FBG des différentes Ténascines étaient capable d’induire une transitionépithélio-mésenchymateuse ainsi qu’une cytostase dans les cellules épithéliales,deux phénomènes classiquement induits par le TGF-Beta bioactif. Enfin, nous avons débuté un second projet visant à caractériser les variants du domaine FBG-Xretrouvés chez des patients souffrants de Syndrome D’Ehlers-Danlos « classicallike». Cette étude vise à identifier l’impact de ces variants sur la capacité de la TN-Xà activer le TGF-Beta latent.

Cancer is a systemic disease involving multiple components produced from both tumor cells themselves and surrounding stromal cells. The pro- or anti-tumoral role of the stroma is still under debate. Indeed, it has long been considered the main physical barrier to the diffusion of chemotherapy by its dense and fibrous nature and its poor vascularization. However, in murine models, the depletion of fibroblasts, the main ExtraCellular Matrix (ECM)-producing cells, led to more aggressive tumors even though they were more susceptible to anti-angiogenic and immuno-modulators. Tenascin-C (TNC) is a multifunctional matricellular glycoprotein (i.e. an ECM protein also able to induce signaling pathway) and is considered as a marker of tumor expansion and metastasis. However, the status of other tenascin (TN) family members and particularly Tenascin-X (TNX) has been far less studied during this pathological process and is still controversial. Herein, through (1) in silico analyses of the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases and (2) immunohistochemistry staining of Tissue MicroArrays (TMA), we performed a large and extensive study of TNX expression at both mRNA and protein levels (1) in the 6 cancers with the highest incidence and mortality in the world (i.e. lung, breast, colorectal, prostate, stomach and liver) and (2) in the cancers for which sparse data regarding TNX expression already exist in the literature. We thus demonstrated that, in most cancers, TNX expression is significantly downregulated during cancer progression and we also highlighted, when data were available, that high TNXB mRNA expression in cancer is correlated with a good survival prognosis.

Resume Les robots mobiles ont une autonomie limitee lorsqu'ils se retrouvent en environnement inconnu. Ils doivent alors construire une carte de leur environnement tout en s'y situant en meme temps. Cette carte servira par la suite a la planification de trajectoire pour que le robot puisse effectuer sa tâche. Cette carte globale se doit donc d'etre la plus precise possible, sinon le robot pourrait rencontrer des obstacles nuisibles et ne jamais atteindre la destination desiree. Le processus pour obtenir cette carte est compose d'une premiere phase d'acquisition ou le robot utilise un LIDAR lui permettant de modeliser son environnement immediat par un nuage de points. Ensuite, il faut relier les acquisitions entre elles le plus fidelement possible pour obtenir un nuage de points couvrant une plus grande partie de l'environnement. Les balayages sont d'abord regroupes deux par deux par un algorithme de recalage appele ICP. Cette technique est tres efficace lorsque l'estime initial de la position est pres du resultat recherche. Par contre, lorsque ces conditions initiales sont plus erronees, il est fortement possible de converger vers un minimum local. Cette erreur de recalage se propage ensuite lorsque le robot continue sa trajectoire. Il est donc important de trouver une technique permettant de qualifier le resultat d'un recalage et ainsi de rejeter les mauvaises informations. La metrique proposee dans ce travail est un algorithme de discrimination ou plusieurs parametres sont compares entre eux. Une base de donnees composee de plusieurs bons et mauvais resultats separes en deux groupes respectifs a ete creee. Elle permet ensuite de comparer un nouveau recalage par rapport a ces deux groupes et d'attribuer une probabilite d'appartenance a chacun des groupes. Ces valeurs donnent alors une indication de la fiabilite du recalage. Les resultats obtenus donnent un taux d'erreur de classification de 2.12%. Cette metrique est par la suite utilisee pour corriger l'accumulation d'erreurs au cours d'une longue trajectoire. En effet, lorsque le robot revisite un endroit deja modelise, il obtient de nouvelles informations permettant d'effectuer une correction sur sa position estimee et ainsi de reduire l'erreur accumulee. Ceci est fait a l'aide d'un algorithme de distribution d'erreur ou le lien entre chaque paire de balayages est modelise par des ressorts. L'ensemble des ressorts et des balayages constitue alors un systeme dynamique qui aura tendance a converger vers une position stable qui correspond au minimum d'energie. Les positions estimees des balayages seront alors modifiees et l'erreur accumulee sera corrigee pour obtenir une carte globale de l'environnement qui est plus uniforme. La metrique de recalage permet de determiner les constantes de raideurs des ressorts. Ainsi, les recalages moins fiables seront representes par des ressorts plus flexibles qui pourront s'etirer pour mieux respecter les contraintes des ressorts plus rigides correspondant aux bons recalages.----------Abstract Mobile robots have a limited autonomy when they are placed in an unknown environment. They must simultaneously create a map and position themself on that map. Then the robot will be able to plan his trajectory to avoid obstacles and accomplish its task. The global map must be precise, otherwise the robot could hit an obstacle or get lost and not reach his destination. The first step to obtain this global map is to use a LIDAR to scan the immediate environment and modelize it by a scatter plot. Then, scans are registered together to create a bigger scatter plot and so on. The scans are first registered two by two with a registration algorithm called ICP. This algorithm leads to a very good estimate of the position if the initial conditions are good enough. Otherwise, it may converge towards a local minima and introduce an error in the global map. This error is then propagated along the robot path. Thus, it is very important to qualify the registration result to be able to reject false informations. This work introduces a discrimination algorithm which compares specific parameters to obtain a registration metric. A database composed of multiple registration results has been created. It is composed of two distinct groups which are the good and the bad results. Then, a new registration result can be compared against these groups to see which ones it relates the most to. The probability of appartenance of this new data to each group gives the probability that the registration converged toward the solution. The parameters used in the discrimination algorithm give an apparent error rate of 2.12%.This metric is then used to minimize the error accumulation during long trajectories. When the robot reaches a previously visited location, it can acquire new data and this new information can be used to correct its estimated position and reduce the errors accumulated during its journey. An error distribution error is used to make this correction. Virtual springs are connected between each pair of scans. Then, the springs and scans constitute a dynamic system which naturally tends towards a stable position consisting of an energy minimum. The new scans position consist of a more consistent map where the error is equally distributed. The registration metric can be used to assign the stiffness of the springs. A less reliable registration result leads to a less stiff spring which can stretch to meet the constraints of the other springs representing better registration results.