Clusterin overexpression in mice exacerbates diabetic phenotypes but suppresses tumor progression in a mouse melanoma model.

Clusterin (CLU) is an ATP-independent small heat shock protein-like chaperone, which functions both intra- and extra-cellularly. Consequently, it has been functionally involved in several physiological (including aging), as well as in pathological conditions and most age-related diseases, e.g., cancer, neurodegeneration, and metabolic syndrome. To address CLU function at an in vivo model we established CLU transgenic (Tg) mice bearing ubiquitous or pancreas-targeted CLU overexpression (OE). Our downstream analyses in established Tg lines showed that ubiquitous or pancreas-targeted CLU OE in mice affected antioxidant, proteostatic and metabolic pathways. Targeted OE of CLU in the pancreas, which also resulted in CLU upregulation in the liver likely via systemic effects, increased basal glucose levels in the circulation and exacerbated diabetic phenotypes. Furthermore, by establishing a syngeneic melanoma mouse tumor model we found that ubiquitous CLU OE suppressed melanoma cells growth, indicating a likely tumor suppressor function in early phases of tumorigenesis. Our observations provide in vivo evidence corroborating the notion that CLU is a potential modulator of metabolic and/or proteostatic pathways playing an important role in diabetes and tumorigenesis.

Antitumor Reactive T-Cell Responses Are Enhanced In Vivo by DAMP Prothymosin Alpha and Its C-Terminal Decapeptide.

Prothymosin α (proTα) and its C-terminal decapeptide proTα(100-109) were shown to pleiotropically enhance innate and adaptive immune responses. Their activities have been broadly studied in vitro, focusing primarily on the restoration of the deficient immunoreactivity of cancer patients' leukocytes. Previously, we showed that proTα and proTα(100-109) act as danger-associated molecular patterns (DAMPs), ligate Toll-like receptor-4, signal through TRIF- and MyD88-dependent pathways, promote the maturation of dendritic cells and elicit T-helper type 1 (Th1) immune responses in vitro, leading to the optimal priming of tumor antigen-reactive T-cell functions. Herein, we assessed their activity in a preclinical melanoma model. Immunocompetent mice bearing B16.F1 tumors were treated with two cycles of proTα or proTα(100-109) together with a B16.F1-derived peptide vaccine. Coadministration of proTα or proTα(100-109) and the peptide vaccine suppressed melanoma-cell proliferation, as evidenced by reduced tumor-growth rates. Higher melanoma infiltration by CD3+ T cells was observed, whereas ex vivo analysis of mouse total spleen cells verified the in vivo induction of melanoma-reactive cytotoxic responses. Additionally, increased levels of proinflammatory and Th1-type cytokines were detected in mouse serum. We propose that, in the presence of tumor antigens, DAMPs proTα and proTα(100-109) induce Th1-biased immune responses in vivo. Their adjuvant ability to orchestrate antitumor immunoreactivities can eventually be exploited therapeutically in humans.

Mapping Interactome Networks of DNAJC11, a Novel Mitochondrial Protein Causing Neuromuscular Pathology in Mice.

We previously identified DNAJC11, a mitochondrial outer membrane protein of unknown function, as a novel genetic cause in modeled neuromuscular disease. To understand the physiological role of DNAJC11, we employed a proteomic approach for the identification of the DNAJC11 interactome, through the expression of DNAJC11-FLAG in HEK293FT cells and transgenic mice. Our analysis confirmed known DNAJC11-interacting proteins including members of the MICOS complex that organize mitochondrial cristae formation. Moreover, we identified in both biological systems novel mitochondrial interactions including VDACs that exchange metabolites across the outer mitochondrial membrane. In HEK293FT cells, DNAJC11 preferentially interacted with ribosomal subunits and chaperone proteins including Hsp70 members, possibly correlating DNAJC11 with cotranslational folding and import of mitochondrial proteins in metabolically active cells. Instead, the DNAJC11 interactome in the mouse cerebrum was enriched for synaptic proteins, supporting the importance of DNAJC11 in synapse and neuronal integrity. Moreover, we demonstrated that the DUF3395 domain is critically involved in DNAJC11 protein-protein interactions, while the J-domain determines its mitochondrial localization. Collectively, these results provide a functional characterization for DNAJC11 domains, while the identified interactome networks reveal an emerging role of DNAJC11 in mitochondrial biogenesis and response to microenvironment changes and requirements.

Oxygen and Glucose Deprivation Alter Synaptic Distribution of Tau Protein: The Role of Phosphorylation.

Alterations in tau synaptic distribution are considered to underlie synaptic dysfunction observed in Alzheimer's disease (AD). In the present study, brain blood hypoperfusion was simulated in mouse brain slices, and tau levels and phosphorylation were investigated in total extracts, as well as in postsynaptic density fractions (PSDs) and non-PSDs obtained through differential extraction and centrifugation. Oxygen deprivation (OD) resulted in tau dephosphorylation at several AD-related residues and activation of GSK3ß and phosphatase PP2A. On the contrary, glucose deprivation (GD) did not affect total levels of cellular tau or its phosphorylation despite inactivation of GSK3ß. However, tau distribution in PSD and non-PSD fractions and the pattern of tau phosphorylation in these compartments is highly complex. In PSDs, tau was increased under GD conditions and decreased under OD conditions. GD resulted in tau dephosphorylation at Ser199, Ser262, and Ser396 while OD resulted in tau hyperphosphorylation at Ser199 and Ser404. In the non-PSD fraction, GD or OD resulted in lower levels of tau, but the phosphorylation status of tau was differentially affected. In GD conditions, tau was found dephosphorylated at Ser199, Thr205, and Ser404 and hyperphosphorylated at Ser262. However, in OD conditions tau was found hyperphosphorylated at Thr205, SerSer356, Ser396, and Ser404. Combined OD and GD resulted in degradation of cellular tau and dephosphorylation of PSD tau at Ser396 and Ser404. These results indicate that oxygen deprivation causes dephosphorylation of tau, while GD and OD differentially affect distribution of total tau and tau phosphorylation variants in neuronal compartments by activating different mechanisms.

Inhibition of the neuronal NFκB pathway attenuates bortezomib-induced neuropathy in a mouse model.

Bortezomib is a proteasome inhibitor with a remarkable antitumor activity, used in the clinic as first line treatment for multiple myeloma. One hallmark of bortezomib mechanism of action in neoplastic cells is the inhibition of nuclear factor kappa B (NFκB), a transcription factor involved in cell survival and proliferation. Bortezomib-induced peripheral neuropathy is a dose-limiting toxicity that often requires adjustment of treatment and affects patient's prognosis and quality of life. Since disruption of NFκB pathway can also affect neuronal survival, we assessed the role of NFκB in bortezomib-induced neuropathy by using a transgenic mouse that selectively provides blockage of the NFκB pathway in neurons. Interestingly, we observed that animals with impaired NFκB activation developed significantly less severe neuropathy than wild type animals, with particular preservation of large myelinated fibers, thus suggesting that neuronal NFκB activation plays a positive role in bortezomib induced neuropathy and that bortezomib treatment might induce neuropathy by inhibiting NFκΒ in non-neuronal cell types or by targeting other signaling pathways. Therefore, inhibition of NFκB might be a promising strategy for the cotreatment of cancer and neuropathy.

IFN-ß differentially regulates the function of T cell subsets in MS and EAE.

Multiple sclerosis (MS) is considered as a T cell mediated autoimmune disease of the CNS, although a pathogenic role has also been attributed to other immune cell types as well as to environmental and genetic factors. Considering that T cells are interesting from an immunopathogenic point of view and consequently from a therapeutic perspective, various T cell targeted therapies have been approved for MS. Interferon beta (IFN-ß) is widely used as first-line intervention for modulating T cell responses, although its pleiotropic and multifaceted activities influence its effectiveness on the disease development, with mechanisms that are not yet fully understood. Since different T cell populations, including pro-inflammatory and regulatory T cells, might affect the course of MS, the effects of IFN-ß become even more complex. This review will summarize recent findings regarding the T cell targeted effect of IFN-ß in MS and its animal model EAE, with emphasis on the direct actions of endogenous and exogenous IFN-ß on each T cell subpopulation involved in CNS autoimmunity. Delineating how IFN-ß exerts its action on different T cell types may eventually contribute to the designing of therapeutic strategies aiming to improve the effectiveness of this drug for MS treatment.

IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development.

Although interferon-ß is used as first-line therapy for multiple sclerosis, the cell type-specific activity of type I interferons in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis, remains obscure. In this study, we have elucidated the in vivo immunomodulatory role of type I interferon signaling in T cells during experimental autoimmune encephalomyelitis by use of a novel transgenic mouse, carrying a cd2-ifnar1 transgene on a interferon-α/ß receptor 1 null genetic background, thus allowing expression of the interferon-α/ß receptor 1 and hence, a functional type I interferon receptor exclusively on T cells. These transgenic mice exhibited milder experimental autoimmune encephalomyelitis with reduced T cell infiltration, demyelination, and axonal damage in the central nervous system. It is noteworthy that interferon-ß administration in transgenic mice generated a more pronounced, protective effect against experimental autoimmune encephalomyelitis compared with untreated littermates. In vivo studies demonstrated that before experimental autoimmune encephalomyelitis onset, endogenous type I interferon receptor signaling in T cells led to impaired T-helper 17 responses, with a reduced fraction of CCR6(+) CD4(+) T cells in the periphery. At the acute phase, an increased proportion of interleukin-10- and interferon-γ-producing CD4(+) T cells was detected in the periphery of the transgenic mice, accompanied by up-regulation of the interferon-γ-induced gene Irgm1 in peripheral T cells. Together, these results reveal a hitherto unknown T cell-associated protective role of type I interferon in experimental autoimmune encephalomyelitis that may provide valuable clues for designing novel therapeutic strategies for multiple sclerosis.

In vivo investigation of tolerance and antitumor activity of cisplatin-loaded PLGA-mPEG nanoparticles.

The tolerance of BALB/c mice to different doses of blank and cisplatin-loaded PLGA-mPEG nanoparticles and the in vivo anticancer activity of these nanoparticles on SCID mice xenografted with colorectal adenocarcinoma HT 29 cells were investigated. Nanoparticles with an average size of 150-160 nm and approximately 2% w/w cisplatin content were prepared by a modified emulsification and solvent evaporation method. Normal BALB/c mice tolerated three weekly intravenous injections of a relatively high dose of blank PLGA-mPEG nanoparticles (500 mg/kg, equivalent to about 10mg nanoparticles/mouse) and three weekly intravenous injections of a high dose of nanoparticle-entrapped cisplatin (10 mg/kg). Also, histopathology examination indicated that there were no differences in the kidneys or spleens from animals treated with cisplatin-loaded nanoparticles or blank nanoparticles compared to the untreated control group. A moderate granulation of protoplasm of hepatic cells was observed in the livers from mice treated with cisplatin-loaded nanoparticles and blank nanoparticles, however, both the hepatic lobe and the portal hepatis maintained their normal architecture. The cisplatin-loaded PLGA-mPEG nanoparticles appeared to be effective at delaying tumor growth in HT 29 tumor-bearing SCID mice. The group of mice treated with cisplatin-loaded nanoparticles exhibited higher survival rate compared to the free cisplatin group. The results justify further evaluation of the in vivo antitumor efficacy of the PLGA-mPEG/cisplatin nanoparticles.

Role of astrocytes and chemokine systems in acute TNFalpha induced demyelinating syndrome: CCR2-dependent signals promote astrocyte activation and survival via NF-kappaB and Akt.

Chemotactic factors known as chemokines play an important role in the pathogenesis of multiple sclerosis (MS). Transgenic expression of TNFalpha in the central nervous system (CNS) leads to the development of a demyelinating phenotype (TNFalpha-induced demyelination; TID) that is highly reminiscent of MS. Little is known about the role of chemokines in TID but insights derived from studying this model might extend our current understanding of MS pathogenesis and complement data derived from the classic autoimmune encephalomyelitis (EAE) model system. Here we show that in TID, chemokines and their receptors were significantly increased during the acute phases of disease. Notably, the CCL2 (MCP-1)-CCR2 axis and the closely related ligand-receptor pair CCR1-CCL3 (MIP-1alpha) were among the most up-regulated during disease. On the other hand, receptors like CCR3 and CCR4 were not elevated. This significant increase in the levels of chemokines/receptors correlated with robust immune infiltration of the CNS by inflammatory cells, i.e., macrophages, and immune cells particularly T and B cells. Immunostaining and confocal microscopy, along with in vitro studies revealed that astrocytes were a major source of locally produced chemokines and expressed functional chemokine receptors such as CCR2. Using an in vitro system we demonstrate that expression of CCR2 was functional in astrocytes and that signaling via this receptor lead to activation of NF-kB and Akt and was associated with increased astrocyte survival. Collectively, our data suggests that transgenic murine models of MS are useful to dissect mechanisms of disease and that in these models, up-regulation of chemokines and their receptors may be key determinants in TID.

Cellular FLIP (long isoform) overexpression in T cells drives Th2 effector responses and promotes immunoregulation in experimental autoimmune encephalomyelitis.

Cellular FLIP (c-FLIP) is an endogenous inhibitor of death receptor-induced apoptosis through the caspase 8 pathway. It is an NF-kappaB-inducible protein thought to promote the survival of T cells upon activation, and its down-regulation has been implicated in activation-induced cell death. We have generated transgenic mice overexpressing human c-FLIP long form (c-FLIP(L)) specifically in T cells using the CD2 promoter (TgFLIP(L)). TgFLIP(L) mice exhibit increased IgG1 production upon stimulation by a T cell-dependent Ag and a markedly enhanced contact hypersensitivity response to allergen. In addition to showing augmented Th2-type responses, TgFLIP(L) mice are resistant to the development of myelin oligodendrocyte glycoprotein 35-55 peptide-induced experimental autoimmune encephalomyelitis, a Th1-driven autoimmune disease. In vitro analyses revealed that T cells of TgFLIP(L) mice proliferate normally, but produce higher levels of IL-2 and show preferential maturation of Th2 cytokine-producing cells in response to antigenic stimulation. After adoptive transfer, these (Th2) cells protected wild-type recipient mice from experimental autoimmune encephalomyelitis induction. Our results show that the constitutive overexpression of c-FLIP(L) in T cells is sufficient to drive Th2 polarization of effector T cell responses and indicate that it might function as a key regulator of Th cell differentiation.

Attenuation of inflammatory polyarthritis in TNF transgenic mice by diacerein: comparative analysis with dexamethasone, methotrexate and anti-TNF protocols.

The impact of diacerein, an effective cartilage targeted therapy that is used in patients with osteoarthritis, on the development and progression of chronic inflammatory arthritis was evaluated in a tumor necrosis factor (TNF) transgenic mouse model (Tg197). The response to diacerein at 2, 20, or 60 mg/kg daily, as well as the comparative effects of other antiarthritis drugs including dexamethasone (0.5 mg/kg daily), methotrexate (1 mg/kg three times weekly) and an anti-TNF agent (5 mg/kg weekly), were assessed in the Tg197 mice. Treatment was initiated before the onset of arthritis and was continued for 5 weeks. A significant improvement in clinical symptoms was found in all three diacerein treated groups in comparison with untreated groups. Confirming these data, semiquantitative histopathologic analysis of the hind paws revealed a significant reduction not only in cartilage destruction but also in the extent of synovitis and bone erosion in diacerein treated groups in comparison with untreated groups. At the most effective dose tested (2 mg/kg daily), diacerein inhibited the onset of arthritis in 28% and attenuated the progression of arthritis in 35% of the Tg197 mice. Comparative analyses showed diacerein to be more potent than methotrexate but not as effective as dexamethasone or anti-TNF agents in suppressing the progression of the TNF mediated arthritis in this model. These results indicate that diacerein has a disease modifying effect on the onset and progression of TNF driven chronic inflammatory arthritis, suggesting that the prophylactic or therapeutic potential of diacerein in patients with RA should be further examined.

Functional analysis of an arthritogenic synovial fibroblast.

Increasing attention has been directed towards identifying non-T-cell mechanisms as potential therapeutic targets in rheumatoid arthritis. Synovial fibroblast (SF) activation, a hallmark of rheumatoid arthritis, results in inappropriate production of chemokines and matrix components, which in turn lead to bone and cartilage destruction. We have demonstrated that SFs have an autonomous pathogenic role in the development of the disease, by showing that they have the capacity to migrate throughout the body and cause pathology specifically to the joints. In order to decipher the pathogenic mechanisms that govern SF activation and pathogenic potential, we used the two most prominent methods of differential gene expression analysis, differential display and DNA microarrays, in a search for deregulated cellular pathways in the arthritogenic SF. Functional clustering of differentially expressed genes, validated by dedicated in vitro functional assays, implicated a number of cellular pathways in SF activation. Among them, diminished adhesion to the extracellular matrix was shown to correlate with increased proliferation and migration to this matrix. Our findings support an aggressive role for the SF in the development of the disease and reinforce the perspective of a transformed-like character of the SF.

Induction of inducible nitric oxide synthase, argininosuccinate synthase, and GTP cyclohydrolase I in arthritic joints of human tumor necrosis factor-alpha transgenic mice.

OBJECTIVE: Transgenic mice that express human tumor necrosis factor-alpha (Tg197 h-TNF-alpha) develop polyarthritis at 3 to 4 weeks of age leading to severe joint destruction at 8 to 10 weeks of age. Studies have suggested that inducible nitric oxide synthase (iNOS) activity can modulate the progression of arthritis. We investigated the induction of iNOS together with argininosuccinate synthase (AS) and GTP cyclohydrolase I (GTPCH), 2 of the rate-limiting enzymes for high output NO generation, in the Tg197 h-TNF-alpha transgenic model of arthritis. METHODS: We used 4 and 8-week-old Tg197 h-TNF-alpha transgenic mice and wild-type CBA C57B1/6 control mice to investigate the expression of iNOS with respect to that of AS, GTPCH, and 3-nitrotyrosine by quantitative RT-PCR and immunocytochemistry. Urinary NO metabolites were analyzed using a chemiluminescence assay. RESULTS: Inducible NOS, AS, and GTPCH mRNA was found in all study groups; however, only iNOS mRNA showed a clear increase in 4-week-old Tg197 h-TNF-alpha transgenics in comparison to age matched wild-type controls. Abundant iNOS protein expression was found in macrophages and vascular smooth muscle cells in hyperplastic synovium and pannus. AS expression was found in vascular endothelium and fibroblasts of the inflammatory synovium and pannus. GTPCH immunoreactivity was mostly restricted to macrophages in inflammatory synovium. Localization of 3-nitrotyrosine overlapped with that of iNOS, indicating formation of reactive nitrogen species. Consistent with the high output NO generation, there was a 5-fold increase in urinary NO metabolites in 8-week-old Tg197 h-TNF-alpha transgenic mice. CONCLUSION: We characterized the Tg197 h-TNF-alpha transgenic model of inflammatory arthritis in terms of high output NO-generating pathway, and showed that both AS and GTPCH are intimately associated with inflammatory arthritis. The concomitant induction of AS and GTPCH with that of iNOS suggests that they may be important modulators of arthritis, and that they may represent novel targets for modulation of disease activity.