Suppressor of cytokine signaling 3 (SOCS3) down-regulates several signaling pathways in multiple cell types, and previous data suggest that SOCS3 may shut off cytokine activation at the early stages of liver regeneration (Campbell, J.S., L. Prichard, F. Schaper, J. Schmitz, A. Stephenson-Famy, M.E. Rosenfeld, G.M. Argast, P.C. Heinrich, and N. Fausto. 2001.J. Clin. Invest. 107:1285–1292). We developed Socs3 hepatocyte-specific knockout (Socs3 h-KO) mice to directly study the role of SOCS3 during liver regeneration after a two-thirds partial hepatectomy (PH). Socs3 h-KO mice demonstrate marked enhancement of DNA replication and liver weight restoration after PH in comparison with littermate controls. Without SOCS3, signal transducer and activator of transcription 3 (STAT3) phosphorylation is prolonged, and activation of the mitogenic extracellular signal-regulated kinase 1/2 (ERK1/2) is enhanced after PH. In vitro, we show that SOCS3 deficiency enhances hepatocyte proliferation in association with enhanced STAT3 and ERK activation after epidermal growth factor or interleukin 6 stimulation. Microarray analyses show that SOCS3 modulates a distinct set of genes, which fall into diverse physiological categories, after PH. Using a model of chemical-induced carcinogenesis, we found that Socs3 h-KO mice develop hepatocellular carcinoma at an accelerated rate. By acting on cytokines and multiple proliferative pathways, SOCS3 modulates both physiological and neoplastic proliferative processes in the liver and may act as a tumor suppressor.

MMP-10 is expressed by macrophages and epithelium in response to injury, but its functions in wound repair are unknown. We observed increased collagen deposition and skin stiffness in Mmp10−/− wounds with no difference in collagen expression or re-epithelialization. Increased collagen deposition in Mmp10−/− wounds was accompanied by less collagenolytic activity and reduced expression of specific metallocollagenases, particularly MMP-8 and MMP-13, where MMP-13 was the key collagenase. Ablation and adoptive transfer approaches and cell-based models demonstrated that the MMP-10-dependent collagenolytic activity was a product of alternatively activated (M2) resident macrophages. These data demonstrate a critical role for macrophage MMP-10 in controlling the tissue remodeling activity of macrophages and moderating scar formation during wound repair.

Several members of the matrix metalloproteinase (MMP) family control a range of immune processes, such as leukocyte influx and chemokine activity. Stromelysin-2 (MMP10) is expressed by macrophages in numerous tissues after injury; however, little is known of its function. Here, we report that MMP10 is expressed by macrophages in human lungs from patients with cystic fibrosis and induced in mouse macrophages in response to Pseudomonas aeruginosa infection both in vivo and by isolated resident alveolar and bone marrow-derived macrophages (BMDM). Our data indicates that macrophage MMP10 serves a beneficial function in response to acute infection. Whereas wildtype mice survived infection with minimal morbidity, 50% of Mmp10−/− mice died and all showed sustained weight loss (morbidity). Although bacterial clearance and neutrophil influx did not differ between genotypes, macrophage numbers were about 3-fold greater in infected Mmp10−/− lungs than in wildtypes. Adoptive transfer of wildtype BMDM normalized infection-induced morbidity in Mmp10−/− recipients to wildtype levels demonstrating that the protective effect of MMP10 was due to its production by macrophages. Both in vivo and in cultured alveolar macrophages and BMDM, expression of several M1 macrophage markers was elevated, while M2 markers were reduced in Mmp10−/− tissue and cells. Global gene expression analysis revealed that infection-mediated transcriptional changes persisted in Mmp10−/− BMDM long after they were down-regulated in wildtype cells. These results indicate that MMP10 serves a beneficial role in response to acute infection by moderating the pro-inflammatory response of resident and infiltrating macrophages.

In nonalcoholic fatty liver disease (NAFLD), depletion of hepatic antioxidants may contribute to the progression of steatosis to nonalcoholic steatohepatitis (NASH) by increasing oxidative stress that produces lipid peroxidation, inflammation, and fibrosis. We investigated whether depletion of glutathione (GSH) increases NASH-associated hepatic pathology in mice fed a diet deficient in methionine and choline (MCD diet). Wild-type (wt) mice and genetically GSH-deficient mice lacking the modifier subunit of glutamate cysteine ligase (Gclm null mice), the rate-limiting enzyme for de novo synthesis of GSH, were fed the MCD diet, a methionine/choline-sufficient diet, or standard chow for 21 days. We assessed NASH-associated hepatic pathology, including steatosis, fibrosis, inflammation, and hepatocyte ballooning, and used the NAFLD Scoring System to evaluate the extent of changes. We measured triglyceride levels, determined the level of lipid peroxidation products, and measured by qPCR the expression of mRNAs for several proteins associated with lipid metabolism, oxidative stress, and fibrosis. MCD-fed GSH-deficient Gclm null mice were to a large extent protected from MCD diet-induced excessive fat accumulation, hepatocyte injury, inflammation, and fibrosis. Compared with wt animals, MCD-fed Gclm null mice had much lower levels of F2-isoprostanes, lower expression of acyl-CoA oxidase, carnitine palmitoyltransferase 1a, uncoupling protein-2, stearoyl-coenzyme A desaturase-1, transforming growth factor-β, and plas-minogen activator inhibitor-1 mRNAs, and higher activity of catalase, indicative of low oxidative stress, inhibition of triglyceride synthesis, and lower expression of profibrotic proteins. Global gene analysis of hepatic RNA showed that compared with wt mice, the livers of Gclm null mice have a high capacity to metabolize endogenous and exogenous compounds, have lower levels of lipogenic proteins, and increased antioxidant activity. Thus, metabolic adaptations resulting from severe GSH deficiency seem to protect against the development of steatohepatitis.

Partial hepatectomy (PH) consistently results in an early increase of circulating interleukin- 6 (IL-6), which is thought to play a major role in liver regeneration. Activation of this cytokine after PH requires the adaptor protein, MyD88, but the specific MyD88-related receptors involved remain unidentified. It is also unknown whether the magnitude of IL-6 elevation determines the extent of subsequent hepatocyte proliferation. Here, we uncovered artifacts in the assessment of circulating IL-6 levels when using cardiac puncture in mice after PH. By using retro-orbital bleed sampling, we show that the circulating levels of IL-6 after PH were not directly correlated with the extent of hepatocyte DNA synthesis in individual mice. The IL-6 increase after PH was attenuated in all lipopolysaccharide-hyporesponsive mouse strains studied (e.g., C3H/HeJ, Tlr4 null, Cd14 null, Tlr2,4,9 null, and Tlr2,4-Caspase1 null) and was severely abrogated in Myd88 null mice. Despite attenuated IL-6 levels, Tlr4 null mice showed normal signaling downstream of IL-6 and normal hepatocyte proliferation. In contrast, Myd88 null mice showed severe impairments in signal transducer and activator of transcription 3 phosphorylation and Socs3 induction, but had enhanced and prolonged extracellular signal-related kinase 1 and 2 phosphorylation in the first 6 hours after PH. Unexpectedly, these changes were associated with accelerated initiation of hepatocyte proliferation, as assessed by hepatocyte bromodeoxyuridine incorporation, phospho-histone H3 immunostaining, and cyclin E and A protein expression. Conclusion TLR-4 signaling contributes to IL-6 activation after PH, but the Tlr4-independent component appears sufficient for ensuring intact signaling downstream of IL-6. The lack of correlation between IL-6 levels and hepatocyte proliferation after PH, and the accelerated start of hepatocyte proliferation in Myd88 null mice despite abrogated cytokine activation, may highlight relevant antiproliferative effects of IL-6 signaling, possibly via Socs3, in the regulation of liver regeneration.

Macrophages have important functional roles in regulating the timely promotion and resolution of inflammation. While many of the intracellular signaling pathways involved in the pro-inflammatory responses of macrophages are well characterized, the components that regulate macrophage reparative properties are less well understood. We identified the MEK1/2 pathway as a key regulator of macrophage reparative properties. Pharmacological inhibition of the MEK1/2 pathway (MEKi) significantly increased expression of IL-4/IL-13 (M2) responsive genes in murine bone marrow-derived and alveolar macrophages. Deletion of the MEK1 gene using LysMCre+/+MEK1fl/fl macrophages as an alternate approach yielded similar results. MEKi enhanced STAT6 phosphorylation, and MEKi induced changes in M2 polarization were dependent on STAT6. In addition, MEKi-treatment significantly increased both murine and human macrophage efferocytosis of apoptotic cells (AC) independent of macrophage polarization and STAT6. These phenotypes were associated with increased gene and protein expression of Mertk, Tyro3, and Abca1, three proteins that promote macrophage efferocytosis. We also studied the effects of MEKi on in vivo macrophage efferocytosis and polarization. MEKi treated mice had increased efferocytosis of apoptotic PMNs instilled into the peritoneum. Furthermore, administration of MEKi after LPS-induced lung injury led to improved recovery of weight, fewer neutrophils in the alveolar compartment, and greater macrophage M2 polarization. Collectively, these results show that MEK1/2 inhibition is capable of promoting reparative properties of both murine and human macrophages. These studies suggest that the MEK1/2 pathway may be a therapeutic target to promote the resolution of inflammation via modulation of macrophage functions.

Background: Macrophage plasticity allows cells to adopt different phenotypes, a property with important implications in disorders such as cystic fibrosis (CF) and asthma. Objective: We sought to examine the transcriptional and functional significance of macrophage repolarization from an M1 to an M2 phenotype and assess the role of a common human genetic disorder (CF) and a prototypical allergic disease (asthma) in this transformation. Methods: Monocyte-derived macrophages were collected from healthy subjects and patients with CF and polarized to an M2 state by using IL-4, IL-10, glucocorticoids, apoptotic PMNs, or azithromycin. We performed transcriptional profiling and

A disintegrin and metalloproteinase 17 (ADAM17), or tumor necrosis factor (TNF)-α-converting enzyme, is a key metalloproteinase and physiological convertase for a number of putative targets that play critical roles in cytokine and growth factor signaling. These interdependent pathways are essential components of the signaling network that links liver function with the compensatory growth that occurs during liver regeneration following 2/3 partial hepatectomy (PH) or chemically induced hepatotoxicity. Despite identification of many soluble factors needed for efficient liver regeneration, very little is known about how such ligands are regulated in the liver. To directly study the role of ADAM17 in the liver, we employed two cell-specific ADAM17 knockout (KO) mouse models. Using lipopolysaccharide (LPS) as a robust stimulus for TNF release, we found attenuated levels of circulating TNF in myeloid-specific ADAM17 KO mice (ADAM17 m-KO) and, unexpectedly, in mice with hepatocyte-specific ADAM17 deletion (ADAM17 h-KO), indicating that ADAM17 expression in both cell types plays a role in TNF shedding. After 2/3 PH, induction of TNF, TNFR1, and amphiregulin (AR) was significantly attenuated in ADAM17 h-KO mice, implicating ADAM17 as the primary sheddase for these factors in the liver. Surprisingly, the extent and timing of hepatocyte proliferation were not affected after PH or carbon tetrachloride injection in ADAM17 h-KO or ADAM17 m-KO mice. We conclude that ADAM17 regulates TNF, TNFR1, and AR in the liver, and its expression in both hepatocytes and myeloid cells is important for TNF regulation after LPS injury or 2/3 PH, but is not required for liver regeneration.