Mounting an effective immune response, while also protecting tissue integrity, is critical for host survival. We used a combined genomic and proteomic approach to investigate the role of extracellular matrix (ECM) proteolysis in achieving this balance in the lung during influenza virus infection. We identified the membrane-tethered matrix metalloprotease MT1-MMP as a prominent host-ECM-remodeling collagenase in influenza infection. Selective inhibition of MT1-MMP protected the tissue from infection-related structural and compositional tissue damage. MT1-MMP inhibition did not significantly alter the immune response or cytokine expression. The available flu therapeutic Oseltamivir did not prevent lung ECM damage and was less effective than anti-MT1-MMP in influenza virus Streptococcus pneumoniae coinfection paradigms. Combination therapy of Oseltamivir with anti-MT1-MMP showed a strong synergistic effect and resulted in complete recovery of infected mice. This study highlights the importance of tissue resilience in surviving infection and the potential of such host-pathogen therapy combinations for respiratory infections.

Abnormal architectures of collagen fibers in the extracellular matrix (ECM) are hallmarks of many invasive diseases, including cancer. Targeting specific stages of collagen assembly in vivo presents a great challenge due to the involvement of various crosslinking enzymes in the multistep, hierarchical process of ECM build-up. Using advanced microscopic tools, we monitored stages of fibrillary collagen assembly in a native fibroblast-derived 3D matrix system and identified anti-lysyl oxidase-like 2 (LOXL2) antibodies that alter the natural alignment and width of endogenic fibrillary collagens without affecting ECM composition. The disrupted collagen morphologies interfered with the adhesion and invasion properties of human breast cancer cells. Treatment of mice bearing breast cancer xenografts with the inhibitory antibodies resulted in disruption of the tumorigenic collagen superstructure and in reduction of primary tumor growth. Our approach could serve as a general methodology to identify novel therapeutics targeting fibrillary protein organization to treat ECMassociated pathologies.

BackgroundAmyotrophic lateral sclerosis (ALS) is a motor neuron (MN) disease characterized by the loss of MNs in the central nervous system. As MNs die, patients progressively lose their ability to control voluntary movements, become paralyzed and eventually die from respiratory/deglutition failure. Despite the selective MN death in ALS, there is growing evidence that malfunctional astrocytes play a crucial role in disease progression. Thus, transplantation of healthy astrocytes may compensate for the diseased astrocytes.MethodsWe developed a good manufacturing practice-grade protocol for generation of astrocytes from human embryonic stem cells (hESCs). The first stage of our protocol is derivation of astrocyte progenitor cells (APCs) from hESCs. These APCs can be expanded in large quantities and stored frozen as cell banks. Further differentiation of the APCs yields an enriched population of astrocytes with more than 90% GFAP expression (hES-AS). hES-AS were injected intrathecally into hSOD1G93A transgenic mice and rats to evaluate their therapeutic potential. The safety and biodistribution of hES-AS were evaluated in a 9-month study conducted in immunodeficient NSG mice under good laboratory practice conditions.ResultsIn vitro, hES-AS possess the activities of functional healthy astrocytes, including glutamate uptake, promotion of axon outgrowth and protection of MNs from oxidative stress. A secretome analysis shows that these hES-AS also secrete several inhibitors of metalloproteases as well as a variety of neuroprotective factors (e.g. TIMP-1, TIMP-2, OPN, MIF and Midkine). Intrathecal injections of the hES-AS into transgenic hSOD1G93A mice and rats significantly delayed disease onset and improved motor performance compared to sham-injected animals. A safety study in immunodeficient mice showed that intrathecal transplantation of hES-AS is safe. Transplanted hES-AS attached to the meninges along the neuroaxis and survived for the entire duration of the study without formation of tumors or teratomas. Cell-injected mice gained similar body weight to the sham-injected group and did not exhibit clinical signs that could be related to the treatment. No differences from the vehicle control were observed in hematological parameters or blood chemistry.ConclusionOur findings demonstrate the safety and potential therapeutic benefits of intrathecal injection of hES-AS for the treatment of ALS.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-0890-5) contains supplementary material, which is available to authorized users.

It is well established that the expression profiles of multiple and possibly redundant matrix-remodeling proteases (e.g., collagenases) differ strongly in health, disease, and development. Although enzymatic redundancy might be inferred from their close similarity in structure, their in vivo activity can lead to extremely diverse tissueremodeling outcomes. We observed that proteolysis of collagen-rich natural extracellular matrix (ECM), performed uniquely by individual homologous proteases, leads to distinct events that eventually affect overall ECM morphology, viscoelastic properties, and molecular composition. We revealed striking differences in the motility and signaling patterns, morphology, and gene-expression profiles of cells interacting with natural collagen-rich ECM degraded by different collagenases. Thus, in contrast to previous notions, matrix-remodeling systems are not redundant and give rise to precise ECM-cell crosstalk. Because ECM proteolysis is an abundant biochemical process that is critical for tissue homoeostasis, these results improve our fundamental understanding its complexity and its impact on cell behavior.T he function and integrity of the extracellular matrix (ECM) is vital for cell behavior as well as for whole-tissue homeostasis (1-4). The ECM undergoes constant remodeling during health and disease states. Its components are regularly being deposited, degraded, or otherwise modified. The highly stable fibrillar collagen type I (Col I) is abundant in many organ-derived ECMs and connective tissues (5, 6); it serves as a tissue scaffold, determining ECM mechanical properties and anchoring other ECM proteins necessary for cell function (7). These processes are orchestrated by multiple remodeling enzymes, among which the matrix metalloproteinase (MMP) family plays an important role. Only a few members of this proteinase family, the collagenases, are able to degrade the resistant fibrillar collagens, i.e., Col I, as well as other ECM molecules (8, 9). The collagenases have conserved amino acids in their zinc-containing catalytic domain (8, 10) and display high structural similarities, as reflected by their functional domain organization. Nevertheless, the complex effects exerted by different MMPs on ECM and cells in vivo remain poorly understood.The enzymatic activity of MMPs, and specifically of collagenases, in vivo is tightly regulated (11), with enzymatic dysregulation causing irreversible damage associated with a variety of diseases. Abnormally elevated levels of MMP-1 or of both MMP-1 and MMP-13 have been associated with different types of cancers and with inflammatory diseases (12-21).Here, we explored the degradation patterns of natural collagenrich ECM by two homologous MMPs and tested the response of cells to intact vs. selectively degraded ECM. We collectively profiled the unique remodeling events caused by two secreted collagenases, MMP-1 and MMP-13, by using biochemical, biophysical, and proteomics tools. We show that these proteases drive morphological, biochemical, and visc...

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