Tariq Sethi

Extracellular matrix proteins protect small cell lung cancer cells against apoptosis: a mechanism for small cell lung cancer growth and drug resistance in vivo.

Resistance to chemotherapy is a principal problem in the treatment of small cell lung cancer (SCLC). We show here that SCLC is surrounded by an extensive stroma of extracellular matrix (ECM) at both primary and metastatic sites. Adhesion of SCLC cells to ECM enhances tumorigenicity and confers resistance to chemotherapeutic agents as a result of β1 integrin-stimulated tyrosine kinase activation suppressing chemotherapy-induced apoptosis. SCLC may create a specialized microenvironment, and the survival of cells bound to ECM could explain the partial responses and local recurrence of SCLC often seen clinically after chemotherapy. Strategies based on blocking β1 integrin-mediated survival signals may represent a new therapeutic approach to improve the response to chemotherapy in SCLC.

Galectin-3 expression and secretion links macrophages to the promotion of renal fibrosis.

Macrophages have been proposed as a key cell type in the pathogenesis of renal fibrosis; however, the mechanism by which macrophages drive fibrosis is still unclear. We show that expression of galectin-3, a beta-galactoside-binding lectin, is up-regulated in a mouse model of progressive renal fibrosis (unilateral ureteric obstruction, UUO), and absence of galectin-3 protects against renal myofibroblast accumulation/activation and fibrosis. Furthermore, specific depletion of macrophages using CD11b-DTR mice reduces fibrosis severity after UUO demonstrating that macrophages are key cells in the pathogenesis of renal fibrosis. Disruption of the galectin-3 gene does not affect macrophage recruitment after UUO, or macrophage proinflammatory cytokine profiles in response to interferon-gamma/lipopolysaccharide. In addition, absence of galectin-3 does not affect transforming growth factor-beta expression or Smad 2/3 phosphorylation in obstructed kidneys. Adoptive transfer of wild-type but not galectin-3(-/-) macrophages did, however, restore the fibrotic phenotype in galectin-3(-/-) mice. Cross-over experiments using wild-type and galectin-3(-/-) macrophage supernatants and renal fibroblasts confirmed that secretion of galectin-3 by macrophages is critical in the activation of renal fibroblasts to a profibrotic phenotype. Therefore, we demonstrate for the first time that galectin-3 expression and secretion by macrophages is a major mechanism linking macrophages to the promotion of renal fibrosis.

Regulation of Alternative Macrophage Activation by Galectin-3

Alternative macrophage activation is implicated in diverse disease pathologies such as asthma, organ fibrosis, and granulomatous diseases, but the mechanisms underlying macrophage programming are not fully understood. Galectin-3 is a carbohydrate-binding lectin present on macrophages. We show that disruption of the galectin-3 gene in 129sv mice specifically restrains IL-4/IL-13-induced alternative macrophage activation in bone marrow-derived macrophages in vitro and in resident lung and recruited peritoneal macrophages in vivo without affecting IFN-γ/LPS-induced classical activation or IL-10-induced deactivation. IL-4-mediated alternative macrophage activation is inhibited by siRNA-targeted deletion of galectin-3 or its membrane receptor CD98 and by inhibition of PI3K. Increased galectin-3 expression and secretion is a feature of alternative macrophage activation. IL-4 stimulates galectin-3 expression and release in parallel with other phenotypic markers of alternative macrophage activation. By contrast, classical macrophage activation with LPS inhibits galectin-3 expression and release. Galectin-3 binds to CD98, and exogenous galectin-3 or cross-linking CD98 with the mAb 4F2 stimulates PI3K activation and alternative activation. IL-4-induced alternative activation is blocked by bis-(3-deoxy-3-(3-methoxybenzamido)-β-D-galactopyranosyl) sulfane, a specific inhibitor of extracellular galectin-3 carbohydrate binding. These results demonstrate that a galectin-3 feedback loop drives alternative macrophage activation. Pharmacological modulation of galectin-3 function represents a novel therapeutic strategy in pathologies associated with alternatively activated macrophages.

Complementation of dominant suppression implicates CD98 in integrin activation

The integrin family of adhesion receptors are involved in cell growth, migration and tumour metastasis. Integrins are heterodimeric proteins composed of an α and a β subunit, each with a large extracellular, a single transmembrane, and a short cytoplasmic domain. The dynamic regulation of integrin affinity for ligands in response to cellular signals is central to integrin function. This process is energy dependent and is mediated through integrin cytoplasmic domains. However, the cellular machinery regulating integrin affinity remains poorly understood. Here we describe a genetic strategy to disentangle integrin signalling pathways. Dominant suppression occurs when overexpression of isolated integrin β1 cytoplasmic domains blocks integrin activation. Proteins involved in integrin signalling were identified by their capacity to complement dominant suppression in an expression cloning scheme. CD98, an early T-cell activation antigen that associates with functional integrins, was found to regulate integrin activation. Furthermore, antibody-mediated crosslinking of CD98 stimulated β1 integrin-dependent cell adhesion. These data indicate that CD98 is involved in regulating integrin affinity, and validate an unbiased genetic approach to analysing integrin signalling pathways.

The regulation of inflammation by galectin-3.

Summary:  Galectin‐3 is a β‐galactoside‐binding animal lectin of appro‐ ximately 30 kDa and is evolutionarily highly conserved. Galectin‐3 is promiscuous, its localization within the tissue micro‐environment may be extracellular, cytoplasmic, or nuclear, and it has a concentration‐dependent ability to be monomeric or form oligomers. These properties impart great flexibility on galectin‐3 as a specific regulator of many biological systems including inflammation. For example, in acute tissue damage galectin‐3 is a key component in the host defense against microbes such as Streptococcus pneumoniae. However, if tissue injury becomes repetitive galectin‐3 also appears to be intimately involved in the transition to chronic inflammation, facilitating the walling off of tissue injury with fibrogenesis and organ scarring. Therefore galectin‐3 can be viewed as a regulatory molecule acting at various stages along the continuum from acute inflammation to chronic inflammation and tissue fibrogenesis. In this review, we examine the role of galectin‐3 in inflammation, and discuss the manipulation of galectin‐3 expression as a potentially novel therapeutic strategy in the treatment of a broad range of inflammatory diseases.

Ly6Chi Monocytes Direct Alternatively Activated Profibrotic Macrophage Regulation of Lung Fibrosis

RATIONALE Idiopathic pulmonary fibrosis (IPF) is a devastating disease. Antiinflammatory therapies, including corticosteroids, are of no benefit. The role of monocytes and macrophages is therefore controversial. OBJECTIVES To define the role of monocytes and macrophages during lung fibrogenesis and resolution, and explore the phenotype of the cells involved. METHODS We used multiple in vivo depletional strategies, backed up by adoptive transfer techniques. Further studies were performed on samples from patients with IPF. MEASUREMENTS AND MAIN RESULTS Depletion of lung macrophages during fibrogenesis reduced pulmonary fibrosis as measured by lung collagen (P = 0.0079); fibrosis score (P = 0.0051); and quantitative polymerase chain reaction for surrogate markers of fibrosis Col1 (P = 0.0083) and a-smooth muscle actin (P = 0.0349). There was an associated reduction in markers of the profibrotic alternative macrophage activation phenotype, Ym1 (P = 0.0179), and Arginase 1. The alternative macrophage marker CD163 was expressed on lung macrophages from patients with IPF. Depletion of Ly6Chi circulating monocytes reduced pulmonary fibrosis (P = 0.0052) and the number of Ym1- positive alternatively activated lung macrophages (P = 0.0310). Their adoptive transfer during fibrogenesis exacerbated fibrosis (P = 0.0304); however, adoptively transferred CD45.1 Ly6Chi cells were not found in the lungs of recipient CD45.2 mice. CONCLUSIONS We demonstrate the importance of circulating monocytes and lung macrophages during pulmonary fibrosis, and emphasize the importance of the alternatively activated macrophage phenotype. We show that Ly6Chi monocytes facilitate the progression of pulmonary fibrosis, but are not obviously engrafted into lungs thereafter. Finally, we provide empirical data to suggest that macrophages may have a resolution-promoting role during the reversible phase of bleomycin-induced pulmonary fibrosis.

Regulation of Transforming Growth Factor-beta 1-driven Lung Fibrosis by Galectin-3

RATIONALE Idiopathic pulmonary fibrosis (IPF) is a chronic dysregulated response to alveolar epithelial injury with differentiation of epithelial cells and fibroblasts into matrix-secreting myofibroblasts resulting in lung scaring. The prognosis is poor and there are no effective therapies or reliable biomarkers. Galectin-3 is a β-galactoside binding lectin that is highly expressed in fibrotic tissue of diverse etiologies. OBJECTIVES To examine the role of galectin-3 in pulmonary fibrosis. METHODS We used genetic deletion and pharmacologic inhibition in well-characterized murine models of lung fibrosis. Further mechanistic studies were performed in vitro and on samples from patients with IPF. MEASUREMENTS AND MAIN RESULTS Transforming growth factor (TGF)-β and bleomycin-induced lung fibrosis was dramatically reduced in mice deficient in galectin-3, manifest by reduced TGF-β1-induced EMT and myofibroblast activation and collagen production. Galectin-3 reduced phosphorylation and nuclear translocation of β-catenin but had no effect on Smad2/3 phosphorylation. A novel inhibitor of galectin-3, TD139, blocked TGF-β-induced β-catenin activation in vitro and in vivo and attenuated the late-stage progression of lung fibrosis after bleomycin. There was increased expression of galectin-3 in the bronchoalveolar lavage fluid and serum from patients with stable IPF compared with nonspecific interstitial pneumonitis and controls, which rose sharply during an acute exacerbation suggesting that galectin-3 may be a marker of active fibrosis in IPF and that strategies that block galectin-3 may be effective in treating acute fibrotic exacerbations of IPF. CONCLUSIONS This study identifies galectin-3 as an important regulator of lung fibrosis and provides a proof of principle for galectin-3 inhibition as a potential novel therapeutic strategy for IPF.

Critical role of c-jun (NH2) terminal kinase in paracetamol- induced acute liver failure

Background: Acute hepatic failure secondary to paracetamol poisoning is associated with high mortality. C-jun (NH2) terminal kinase (JNK) is a member of the mitogen-activated protein kinase family and is a key intracellular signalling molecule involved in controlling the fate of cells. Aim: To examine the role of JNK in paracetamol-induced acute liver failure (ALF). Methods: A previously developed mouse model of paracetamol poisoning was used to examine the role of JNK in paracetamol-induced ALF. Results: Paracetamol-induced hepatic JNK activation both in human and murine paracetamol hepatotoxicity and in our murine model preceded the onset of hepatocyte death. JNK inhibition in vivo (using two JNK inhibitors with different mechanisms of action) markedly reduced mortality in murine paracetamol hepatotoxicity, with a significant reduction in hepatic necrosis and apoptosis. In addition, delayed administration of the JNK inhibitor was more effective than N-acetylcysteine after paracetamol poisoning in mice. JNK inhibition was not protective in acute carbon tetrachloride-mediated or anti-Fas antibody-mediated hepatic injury, suggesting specificity for the role of JNK in paracetamol hepatotoxicity. Furthermore, disruption of the JNK1 or JNK2 genes did not protect against paracetamol-induced hepatic damage. Pharmacological JNK inhibition had no effect on paracetamol metabolism, but markedly inhibited hepatic tumour necrosis foctor α (TNF α) production after paracetamol poisoning. Conclusions: These data demonstrated a central role for JNK in the pathogenesis of paracetamol-induced liver failure, thereby identifying JNK as an important therapeutic target in the treatment of paracetamol hepatotoxicity.

Human Dendritic Cells Produce TGF-beta 1 under the Influence of Lung Carcinoma Cells and Prime the Differentiation of CD4(+)CD25(+)Foxp3(+) Regulatory T Cells

Dendritic cells (DCs) have a central role in the development of adaptive immune responses, including antitumor immunity. Factors present in the tumor milieu can alter the maturation of DCs and inhibit their capacity to activate T cells. Using gene expression analysis, we found that human DCs increased the expression of TGF-β1 transcripts following culture with human lung carcinoma cells (LCCs). These DCs produced increased amounts of TGF-β1 protein compared with DCs not exposed to tumor cells. LCCs also decreased the expression of CD86 and HLA-DR by immature DCs. Furthermore, LCCs decreased CD86 expression and the production of TNF-α and IL-12 p70 by mature DCs. Moreover, LCCs also converted mature DCs into cells producing TGF-β1. These TGF-β1-producing DCs were poor at eliciting the activation of naive CD4+ T cells and sustaining their proliferation and differentiation into Th1 (IFN-γ+) effectors. Instead, TGF-β1-producing DCs demonstrated an increased ability to generate CD4+CD25+Foxp3+ regulatory T cells that suppress the proliferation of T lymphocytes. These results identify a novel mechanism by which the function of human DCs is altered by tumor cells and contributes to the evasion of the immune response.

Galectin-3 reduces the severity of pneumococcal pneumonia by augmenting neutrophil function

The Gram-positive Streptococcus pneumoniae is the leading cause of community-acquired pneumonia worldwide, resulting in high mortality. Our in vivo studies show that galectin-3(-/-) mice develop more severe pneumonia after infection with S. pneumoniae, as demonstrated by increased bacteremia and lung damage compared to wild-type mice and that galectin-3 reduces the severity of pneumococcal pneumonia in part by augmenting neutrophil function. Specifically, we show that 1) galectin-3 directly acts as a neutrophil-activating agent and potentiates the effect of fMLP, 2) exogenous galectin-3 augments neutrophil phagocytosis of bacteria and delays neutrophil apoptosis, 3) phagocytosis of apoptotic neutrophils by galectin-3(-/-) macrophages is less efficient compared to wild type, and 4) galectin-3 demonstrates bacteriostatic properties against S. pneumoniae in vitro. Furthermore, ad-back of recombinant galectin-3 in vivo protects galectin-3-deficient mice from developing severe pneumonia. Together, these results demonstrate that galectin-3 is a key molecule in the host defense against pneumococcal infection. Therapeutic strategies designed to augment galectin-3 activity may both enhance inflammatory cell function (by directly affecting neutrophil responsiveness and prolonging neutrophil longevity) and have direct bacteriostatic activity, improving clinical outcomes after severe pneumococcal infection.