Tatiana M. Garcia-Bates

Electrophilic Peroxisome Proliferator–Activated Receptor-γ Ligands Have Potent Antifibrotic Effects in Human Lung Fibroblasts

Pulmonary fibrosis is a progressive scarring disease with no effective treatment. Transforming growth factor (TGF)-beta is up-regulated in fibrotic diseases, where it stimulates differentiation of fibroblasts to myofibroblasts and production of excess extracellular matrix. Peroxisome proliferator-activated receptor (PPAR) gamma is a transcription factor that regulates adipogenesis, insulin sensitization, and inflammation. We report here that a novel PPARgamma ligand, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), is a potent inhibitor of TGF-beta-stimulated differentiation of human lung fibroblasts to myofibroblasts, and suppresses up-regulation of alpha-smooth muscle actin, fibronectin, collagen, and the novel myofibroblast marker, calponin. The inhibitory concentration causing a 50% decrease in aSMA for CDDO was 20-fold lower than the endogenous PPARgamma ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15 d-PGJ(2)), and 400-fold lower than the synthetic ligand, rosiglitazone. Pharmacologic and genetic approaches were used to demonstrate that CDDO mediates its activity via a PPARgamma-independent pathway. CDDO and 15 d-PGJ(2) contain an alpha/beta unsaturated ketone, which acts as an electrophilic center that can form covalent bonds with cellular proteins. Prostaglandin A(1) and diphenyl diselenide, both strong electrophiles, also inhibit myofibroblast differentiation, but a structural analog of 15 d-PGJ(2) lacking the electrophilic center is much less potent. CDDO does not alter TGF-beta-induced Smad or AP-1 signaling, but does inhibit acetylation of CREB binding protein/p300, a critical coactivator in the transcriptional regulation of TGF-beta-responsive genes. Overall, these data indicate that certain PPARgamma ligands, and other small molecules with electrophilic centers, are potent inhibitors of critical TGF-beta-mediated profibrogenic activities through pathways independent of PPARgamma. As the inhibitory concentration causing a 50% decrease in aSMA for CDDO is 400-fold lower than that in rosiglitazone, the translational potential of CDDO for treatment of fibrotic diseases is high.

Peroxisome proliferator-activated receptor-γ ligands induce heme oxygenase-1 in lung fibroblasts by a PPARγ-independent, glutathione-dependent mechanism

Oxidative stress plays an important role in the pathogenesis of pulmonary fibrosis. Heme oxygenase-1 (HO-1) is a key antioxidant enzyme, and overexpression of HO-1 significantly decreases lung inflammation and fibrosis in animal models. Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a transcription factor that regulates adipogenesis, insulin sensitization, and inflammation. We report here that the PPARgamma ligands 15d-PGJ2 and 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), which have potent antifibrotic effects in vitro, also strongly induce HO-1 expression in primary human lung fibroblasts. Pharmacological and genetic approaches are used to demonstrate that induction of HO-1 is PPARgamma independent. Upregulation of HO-1 coincides with decreased intracellular glutathione (GSH) levels and can be inhibited by N-acetyl cysteine (NAC), a thiol antioxidant and GSH precursor. Upregulation of HO-1 is not inhibited by Trolox, a non-thiol antioxidant, and does not involve the transcription factors AP-1 or Nrf2. CDDO and 15d-PGJ2 contain an alpha/beta unsaturated ketone that acts as an electrophilic center that can form covalent bonds with free reduced thiols. Rosiglitazone, a PPARgamma ligand that lacks an electrophilic center, does not induce HO-1. These data suggest that in human lung fibroblasts, 15d-PGJ2 and CDDO induce HO-1 via a GSH-dependent mechanism involving the formation of covalent bonds between 15d-PGJ2 or CDDO and GSH. Inhibiting HO-1 upregulation with NAC has only a small effect on the antifibrotic properties of 15d-PGJ2 and CDDO in vitro. These results suggest that CDDO and similar electrophilic PPARgamma ligands may have great clinical potential as antifibrotic agents, not only through direct effects on fibroblast differentiation and function, but indirectly by bolstering antioxidant defenses.

Association between Magnitude of the Virus-Specific Plasmablast Response and Disease Severity in Dengue Patients

Dengue is a globally expanding disease caused by infection with dengue virus (DENV) that ranges from febrile illness to acute disease with serious complications. Secondary infection predisposes individuals to more severe disease, and B lymphocytes may play a role in this phenomenon through production of Ab that enhance infection. To better define the acute B cell response during dengue, we analyzed peripheral B cells from an adult Brazilian hospital cohort with primary and secondary DENV infections of varying clinical severity. Circulating B cells in dengue patients were proliferating, activated, and apoptotic relative to individuals with other febrile illnesses. Severe secondary DENV infection was associated with extraordinary peak plasmablast frequencies between 4 and 7 d of illness, averaging 46% and reaching 87% of B cells, significantly greater than those seen in mild illness or primary infections. On average >70% of IgG-secreting cells in individuals with severe secondary DENV infection were DENV specific. Plasmablasts produced Ab that cross-reacted with heterotypic DENV serotypes, but with a 3-fold greater reactivity to DENV-3, the infecting serotype. Plasmablast frequency did not correlate with acute serum-neutralizing Ab titers to any DENV serotype regardless of severity of disease. These findings indicate that massive expansion of DENV-specific and serotype cross-reactive plasmablasts occurs in acute secondary DENV infection of adults in Brazil, which is associated with increasing disease severity.

Peroxisome proliferator-activated receptor gamma overexpression suppresses growth and induces apoptosis in human multiple myeloma cells.

Purpose: Peroxisome proliferator-activated receptor γ (PPARγ) is a transcription factor that regulates immune and inflammatory responses. Our laboratory has shown that normal and malignant B cells, including multiple myeloma, express PPARγ. Moreover, certain PPARγ ligands can induce apoptosis in multiple myeloma cells. Because PPARγ ligands can also have PPARγ-independent effects, the role of PPARγ in B-cell malignancies remains poorly understood. To further understand the role of PPARγ, we examined the functional consequences of its overexpression in human multiple myeloma. Experimental Design: In the present work, we developed a lentiviral vector for PPARγ gene delivery. We transduced multiple myeloma cells with a lentivirus-expressing PPARγ and studied the involvement of this receptor on cell growth and viability. Results: PPARγ overexpression decreased multiple myeloma cell proliferation and induced spontaneous apoptosis even in the absence of exogenous ligand. These PPARγ-overexpressing cells were dramatically more sensitive to PPARγ ligand-induced apoptosis compared with uninfected or LV-empty-infected cells. Apoptosis was associated with the down-regulation of antiapoptotic proteins X-linked inhibitor of apoptosis protein and myeloid cell leukemia-1 as well as induction of caspase-3 activity. Importantly, PPARγ overexpression-induced cell death was not abrogated by coincubation with bone marrow stromal cells (BMSC), which are known to protect multiple myeloma cells from apoptosis. Additionally, PPARγ overexpression in multiple myeloma or BMSC inhibited both basal and multiple myeloma-induced interleukin-6 production by BMSC. Conclusions: Our results indicate that PPARγ negatively controls multiple myeloma growth and viability in part through inhibition of interleukin-6 production by BMSC. As such, PPARγ is a viable therapeutic target in multiple myeloma.

Peroxisome Proliferator-Activated Receptor γ Ligands Enhance Human B Cell Antibody Production and Differentiation

Protective humoral immune responses critically depend on the optimal differentiation of B cells into Ab-secreting cells. Because of the important role of Abs in fighting infections and in successful vaccination, it is imperative to identify mediators that control B cell differentiation. Activation of B cells through TLR9 by CpG-DNA induces plasma cell differentiation and Ab production. Herein, we examined the role of the peroxisome proliferator-activated receptor (PPAR)γ/RXRα pathway on human B cell differentiation. We demonstrated that activated B cells up-regulate their expression of PPARγ. We also show that nanomolar levels of natural (15-deoxy-Δ12,14-prostaglandin J2) or synthetic (rosiglitazone) PPARγ ligands enhanced B cell proliferation and significantly stimulated plasma cell differentiation and Ab production. Moreover, the addition of GW9662, a specific PPARγ antagonist, abolished these effects. Retinoid X receptor (RXR) is the binding partner for PPARγ and is required to produce an active transcriptional complex. The simultaneous addition of nanomolar concentrations of the RXRα ligand (9-cis-retinoic acid) and PPARγ ligands to CpG-activated B cells resulted in additive effects on B cell proliferation, plasma cell differentiation, and Ab production. Furthermore, PPARγ ligands alone or combined with 9-cis-retinoic acid enhanced CpG-induced expression of Cox-2 and the plasma cell transcription factor BLIMP-1. Induction of these important regulators of B cell differentiation provides a possible mechanism for the B cell-enhancing effects of PPARγ ligands. These new findings indicate that low doses of PPARγ/RXRα ligands could be used as a new type of adjuvant to stimulate Ab production.

Regulation of Lymphocyte Function by PPARγ: Relevance to Thyroid Eye Disease-Related Inflammation

Thyroid eye disease (TED) is an autoimmune condition in which intense inflammation leads to orbital tissue remodeling, including the accumulation of extracellular macromolecules and fat. Disease progression depends upon interactions between lymphocytes and orbital fibroblasts. These cells engage in a cycle of reciprocal activation which produces the tissue characteristics of TED. Peroxisome proliferator-activated receptor-γ (PPARγ) may play divergent roles in this process, both attenuating and promoting disease progression. PPARγ has anti-inflammatory activity, suggesting that it could interrupt intercellular communication. However, PPARγ activation is also critical to adipogenesis, making it a potential culprit in the pathological fat accumulation associated with TED. This review explores the role of PPARγ in TED, as it pertains to crosstalk between lymphocytes and fibroblasts and the development of therapeutics targeting cell-cell interactions mediated through this signaling pathway.

Role of Peroxisome Proliferator-Activated Receptor Gamma and Its Ligands in the Treatment of Hematological Malignancies

Peroxisome proliferator-activated receptor gamma (PPARγ) is a multifunctional transcription factor with important regulatory roles in inflammation, cellular growth, differentiation, and apoptosis. PPARγ is expressed in a variety of immune cells as well as in numerous leukemias and lymphomas. Here, we review recent studies that provide new insights into the mechanisms by which PPARγ ligands influence hematological malignant cell growth, differentiation, and survival. Understanding the diverse properties of PPARγ ligands is crucial for the development of new therapeutic approaches for hematological malignancies.

15‐deoxy‐Δ12,14 prostaglandin J2‐induced heme oxygenase‐1 in megakaryocytes regulates thrombopoiesis

Summary.  Background: Platelet production is an intricate process that is poorly understood. Recently, we demonstrated that the natural peroxisome proliferator‐activated receptor gamma (PPARγ) ligand, 15‐deoxy‐Δ12,14 prostaglandin J2 (15d‐PGJ2), augments platelet numbers by increasing platelet release from megakaryocytes through the induction of reactive oxygen species (ROS). 15d‐PGJ2 can exert effects independent of PPARγ, such as increasing oxidative stress. Heme oxygenase‐1 (HO‐1) is a potent antioxidant and may influence platelet production. Objectives: To further investigate the influence of 15d‐PGJ2 on megakaryocytes and to understand whether HO‐1 plays a role in platelet production. Methods: Meg‐01 cells (a primary megakaryoblastic cell line) and primary human megakaryocytes derived from cord blood were used to examine the effects of 15d‐PGJ2 on HO‐1 expression in megakaryocytes and their daughter platelets. The role of HO‐1 activity in thrombopoiesis was studied using established in vitro models of platelet production. Results and conclusions: 15d‐PGJ2 potently induced HO‐1 protein expression in Meg‐01 cells and primary human megakaryocytes. The platelets produced from these megakaryocytes also expressed elevated levels of HO‐1. 15d‐PGJ2‐induced HO‐1 was independent of PPARγ, but could be replicated using other electrophilic prostaglandins, suggesting that the electrophilic properties of 15d‐PGJ2 were important for HO‐1 induction. Interestingly, inhibiting HO‐1 activity enhanced ROS generation and augmented 15d‐PGJ2‐induced platelet production, which could be attenuated by antioxidants. These new data reveal that HO‐1 negatively regulates thrombopoiesis by inhibiting ROS.

Neurotrophin signaling through tropomyosin receptor kinases contributes to survival and proliferation of non-Hodgkin lymphoma.

OBJECTIVE Neurotrophin receptor signaling has been increasingly recognized as an important factor in the development and progression of a variety of malignancies. In order to analyze the potential contribution of neurotrophin signaling to lymphoma cell survival, we investigated the role of a neurotrophin axis in promoting survival and proliferation of non-Hodgkin lymphoma (NHL) cells. MATERIALS AND METHODS The role of neurotrophins in the survival and proliferation of NHL cells was determined by exposing cells to the Trk-specific inhibitor, K252a, and then performing (3)H-thymidine incorporation and Annexin-V/propidium iodide staining. The involvement of nuclear factor-kappaB (NF-kappaB) in this process was studied using Western blot, electrophoretic mobility shift assay, and immunofluorescence assays. RESULTS Here we demonstrate that both primary NHL cells and diffuse large B-cell lymphoma cell lines express Trk receptors and their neurotrophin ligands. Furthermore, these cells are sensitive to the Trk-specific inhibitor, K252a, as evidenced by the inhibition of proliferation and/or induction of apoptosis. Analysis of the mechanism into the effects of K252a revealed that, in the OCI-LY3 cell line, K252a induced a subnuclear distribution of NF-kappaB resulting in the sequestration of RelA in the nucleolus, thereby inhibiting NF-kappaB-dependent gene transcription. This results in the loss of interleukin-6 production; a known survival-promoting signal for OCI-LY3, as well as many primary diffuse large B-cell lymphomas. CONCLUSION Thus, Trk receptors represent a novel therapeutic target for the treatment of NHL.

Anti-EGFR Targeted Monoclonal Antibody Isotype Influences Antitumor Cellular Immunity in Head and Neck Cancer Patients

Purpose: EGF receptor (EGFR) is highly overexpressed on several cancers and two targeted anti-EGFR antibodies which differ by isotype are FDA-approved for clinical use. Cetuximab (IgG1 isotype) inhibits downstream signaling of EGFR and activates antitumor, cellular immune mechanisms. As panitumumab (IgG2 isotype) may inhibit downstream EGFR signaling similar to cetuximab, it might also induce adaptive immunity. Experimental Design: We measured in vitro activation of cellular components of the innate and adaptive immune systems. We also studied the in vivo activation of components of the adaptive immune system in patient specimens from two recent clinical trials using cetuximab or panitumumab. Results: Both monoclonal antibodies (mAb) primarily activate natural killer (NK) cells, although cetuximab is significantly more potent than panitumumab. Cetuximab-activated neutrophils mediate antibody-dependent cellular cytotoxicity (ADCC) against head and neck squamous cell carcinomas (HNSCC) tumor cells, and interestingly, this effect was FcγRIIa- and FcγRIIIa genotype–dependent. Panitumumab may activate monocytes through CD32 (FcγRIIa); however, monocytes activated by either mAb are not able to mediate ADCC. Cetuximab enhanced dendritic cell (DC) maturation to a greater extent than panitumumab, which was associated with improved tumor antigen cross-presentation by cetuximab compared with panitumumab. This correlated with increased EGFR-specific cytotoxic CD8+ T cells in patients treated with cetuximab compared with those treated with panitumumab. Conclusions: Although panitumumab effectively inhibits EGFR signaling to a similar extent as cetuximab, it is less effective at triggering antitumor, cellular immune mechanisms which may be crucial for effective therapy of HNSCC. Clin Cancer Res; 22(21); 5229–37. ©2016 AACR.