Tissue inhibitor of metalloproteinases 3 (TIMP3) inhibits not only matrix metalloproteinases but also a disintegrin and metalloproteinase domain family members and thus contributes to controlling diverse processes mediated by proteolysis. We used Timp3(-/-) mice to assess the role of this inhibitor in acute lung injury. After bleomycin-induced injury, inflammation, as indicated by the influx of neutrophils in bronchoalveolar lavage (BAL), peaked at 7 days post-injury in the wild-type mice and began to wane thereafter; however, in Timp3(-/-) mice, inflammation persisted up to 28 days. Furthermore, although the level of chemokines in BAL and lung homogenate was similar in both genotypes, BAL from Timp3(-/-) mice 7, 14, and 28 days post-injury had increased neutrophil chemotactic activity compared with wild-type BAL. At day 14, a higher percentage of apoptotic neutrophils were present in wild-type mice compared with Timp3(-/-) mice, further suggesting that TIMP3 constrains continued neutrophil influx. In addition, total matrix metalloproteinase activity was increased in lungs from Timp3(-/-) mice, and treatment of mice with a synthetic inhibitor of metalloproteinases rescued the enhanced neutrophilia phenotype. These data demonstrate that TIMP3 regulates neutrophil influx in the lung following injury through its ability to inhibit metalloproteinase activity and indicates that TIMP3 functions to promote the resolution of inflammation in the lung.

Tissue inhibitor of metalloproteinases-3 (TIMP-3) has emerged as a key mediator of inflammation. Recently, we reported that the resolution of inflammation is impaired in Timp3 2/2 mice after bleomycininduced lung injury. Here, we demonstrate that after LPS instillation (another model of acute lung injury), Timp32/2 mice demonstrate enhanced and persistent neutrophilia, increased numbers of infiltrated macrophages, and delayed weight gain, compared with wild-type (WT) mice. Because macrophages possess broad immune functions and can differentiate into cells that either stimulate inflammation (M1 macrophages) or are immunosuppressive (M2 macrophages), we examined whether TIMP-3 influences macrophage polarization. Comparisons of the global gene expression of unstimulated or LPS-stimulated bone marrow-derived macrophages (BMDMs) from WT and Timp3 2/2 mice revealed that Timp3 2/2 BMDMs exhibited an increased expression of genes associated with proinflammatory (M1) macrophages, including Il6, Il12, Nos2, and Ccl2. Microarray analyses also revealed a baseline difference in gene expression between WT and Timp3 2/2 BMDMs, suggesting altered macrophage differentiation. Furthermore, the treatment of Timp3 2/2 BMDMs with recombinant TIMP-3 rescued this altered gene expression. We also examined macrophage function, and found that Timp3 2/2 M1 cells exhibit significantly more neutrophil chemotactic activity and significantly less soluble Fas ligand-induced caspase-3/7 activity, a marker of apoptosis, compared with WT M1 cells. Macrophage differentiation into immunosuppressive M2 cells is mediated by exposure to IL-4/IL-13, and we found that Timp3 2/2 M2 macrophages demonstrated a lower expression of genes associated with an anti-inflammatory phenotype, compared with WT M2 cells. Collectively, these findings indicate that TIMP-3 functions to moderate the differentiation of macrophages into proinflammatory (M1) cells.Keywords: resolution of inflammation; macrophage; metalloproteinase; lung injury Several immunosuppressive mechanisms have evolved to ensure that acute, generally beneficial inflammation does not progress to chronic, destructive inflammation. Beyond reversal of the initiating event (e.g., bacterial clearance and wound closure), active host processes, such as the release of the anti-inflammatory cytokines IL-4, IL-10, and IL-13, among other processes (1), actively repress the proinflammatory properties of various cell types. Macrophages, through their ability to clear neutrophils and release anti-inflammatory cytokines, are thought to be important in the resolution of inflammation (2).Generally speaking, macrophages are key effectors promoting the shift from a proinflammatory to an anti-inflammatory environment, and can be broadly classified into two groups: classically activated (M1) and alternatively activated (M2) macrophages, which can be further subdivided into specialized M2 groups (3). M1 macrophages are induced by proinflammatory Th1 cytokines, such as IFN-g, and are characterized by the production of proinfl...

CD36 and Glut4 are major transporters of fatty acids and glucose and PPARγ is an important modulator of insulin sensitivity and lipid sensing in skeletal muscle, especially when consuming a high fat diet (HFD). Dysregulation of these transporters and modulators in skeletal muscle may influence the susceptibility to obesity. The purpose of the current experiment was to determine the effects of a HFD on factors associated with glucose and lipid storage and metabolism in the skeletal muscle of obesity‐prone and obesity‐resistant rats. Osborne‐Mendel (OM) and S5B/Pl (S5B) rats were fed a HFD or a low fat diet for 2 weeks. The gastrocnemius muscle was removed and tissue was sliced and stained for oxidative enzymes or protein was isolated for determination of protein expression of CD36, Glut4, and PPARγ using Western Blot. OM rats had a higher proportion of oxidative muscle fibers compared to S5B rats. Additionally, OM rats had a greater expression of CD36 compared to S5B rats, which was independent of diet. The consumption of a HFD reduced Glut4 levels in the skeletal muscle of OM rats compared to S5B rats and OM rats expressed less PPARγ than S5B rats, regardless of diet. Overall, these differences suggest that OM rats consuming a HFD have impaired expression of the main glucose transporter and a key modulator of lipid detection, PPARγ, in skeletal muscle, which may contribute to their susceptibility to obesity.

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