Targeting monocytes/macrophages in fibrosis and cancer diseases: Therapeutic approaches.

Over almost 140 years since their identification, the knowledge about macrophages has unbelievably evolved. The 'big eaters' from being thought of as simple phagocytic cells have been recognized as master regulators in immunity, homeostasis, healing/repair and organ development. Long considered to originate exclusively from bone marrow-derived circulating monocytes, macrophages have been also demonstrated to be the first immune cells colonizing tissues in the developing embryo and persisting in adult life by self-renewal, as long-lived tissue resident macrophages. Therefore, heterogeneous populations of macrophages with different ontogeny and functions co-exist in tissues. Macrophages act as sentinels of homeostasis and are intrinsically programmed to lead the wound healing and repair processes that occur after injury. However, in certain pathological circumstances macrophages get dysfunctional, and impaired or aberrant macrophage activities become key features of diseases. For instance, in both fibrosis and cancer, that have been defined 'wounds that do not heal', dysfunctional monocyte-derived macrophages overall play a key detrimental role. On the other hand, due to their plasticity these cells can be 're-educated' and exert anti-fibrotic and anti-cancer functions. Therefore macrophages represent an important therapeutic target in both fibrosis and cancer diseases. The current review will illustrate new insights into the role of monocytes/macrophages in these devastating diseases and summarize new therapeutic strategies and applications of macrophage-targeted drug development in their clinical setting.

Preventive and therapeutic effects of thymosin ß4 N-terminal fragment Ac-SDKP in the bleomycin model of pulmonary fibrosis.

In this study, the bleomycin model of pulmonary fibrosis was utilized to investigate putative anti-fibrotic activity of Ac-SDKP in vivo. Male CD-1 mice received intra-tracheal bleomycin (BLEO, 1 mg/kg) instillation in the absence or presence of Ac-SDKP (a dose of 0.6 mg/kg delivered intra-peritoneally on the day of BLEO treatment, d0, followed by bi-weekly additional doses). To evaluate therapeutic effects in a subset of mice, Ac-SDKP was administered one week after BLEO instillation (d7). Animals were sacrificed at one, two, or three weeks later. Measurement of fluid and collagen content in the lung, Broncho Alveolar Lavage Fluid (BALF) analysis, lung histology, immunohistochemistry (IHC), and molecular analysis were performed. Compared to BLEO-treated mice, animals that received also Ac-SDKP (at both d0 and d7) had significantly decreased mortality, weight loss, inflammation (edema, and leukocyte lung infiltration), lung damage (histological evidence of lung injury), and fibrosis (collagen histological staining and soluble collagen content in the lung) at up to 21 days. Moreover, IHC and quantitative RT-PCR results demonstrated a significant decrease in BLEO-induced IL-17 and TGF-ß expression in lung tissue. Importantly, α-SMA expression, the hallmark of myofibroblast differentiation, was also decreased. This is the first report showing not only a preventive protective role of Ac-SDKP but also its significant therapeutic effects in the bleomycin model of pulmonary fibrosis, thus supporting further preclinical and clinical studies.

Effects of thymosin ß4 and its N-terminal fragment Ac-SDKP on TGF-ß-treated human lung fibroblasts and in the mouse model of bleomycin-induced lung fibrosis.

UNLABELLED: Thymosin ß4 (Tß4) and its amino-terminal fragment comprising N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) have been reported to act as anti-inflammatory and anti-fibrotic agents in vitro and in vivo. In recent papers, we have shown that Tß4 exerts a widely protective role in mice treated with bleomycin, and in particular, we have demonstrated its inhibitory effects on both inflammation and early fibrosis. OBJECTIVES: In this study, the putative anti-proliferative and anti-fibrogenic effects of Tß4 and Ac-SDKP were evaluated in vitro. In addition, the effects of Tß4 up to 21 days were evaluated in the bleomycin mouse model of lung fibrosis. METHODS: We utilized both control and TGF-ß-stimulated primary human lung fibroblasts isolated from both idiopathic pulmonary fibrosis (IPF) and control tissues. The in vivo effects of Tß4 were assessed in CD1 mice treated with bleomycin. RESULTS: In the in vitro experiments, we observed significant anti-proliferative effects of Ac-SDKP in IPF fibroblasts. In those cells, Ac-SDKP significantly inhibited TGF-ß-induced α-SMA and collagen expression, hallmarks of fibroblast differentiation into myofibroblasts triggered by TGF-ß. In vivo, despite its previously described protective role in mice treated with bleomycin at 7 days, Tß4 failed to prevent fibrosis induced by the drug at 14 and 21 days. CONCLUSION: We conclude that, compared to Tß4, Ac-SDKP may have greater potential as an anti-fibrotic agent in the lung. Further in vivo experiments are warranted.

Protein profile of exhaled breath condensate determined by high resolution mass spectrometry.

A method based on liquid chromatography/high resolution tandem mass spectrometry coupled with electrophoretic separation, for determination and relative quantification of the protein composition of exhaled breath condensate (EBC), was developed. Application of the procedure to a sample of EBC, pooled from nine healthy subjects, resulted in the identification of 167 unique gene products, 113 of which not previously reported in EBC samples. The abundance of the protein identified was estimated by means of the exponentially modified protein abundance index protocol (emPAI). Cytokeratins were by far the most abundant proteins in EBC samples. Many of the identified proteins were associated with multiple cellular location with cytoplasm constituting the largest group. Cytosol, nucleus, membrane, cytoskeleton and extracellular were other abundantly represented locations. No amylase was detected, suggesting the absence of saliva protein contamination. The profile obtained represents the most comprehensive protein characterization of EBC so far reported and demonstrates that this approach provides a powerful tool for investigating the protein profile of EBC samples. Compared with analogous investigations, this study also shows that the protein profile of EBC is strongly affected by the sampling method adopted.

Anti-inflammatory and antifibrotic effects of resveratrol in the lung.

Resveratrol, a natural polyphenolic molecule with several biological activities, is a well recognized anti-oxidant, anti-aging and cancer chemopreventive agent. Moreover, resveratrol anti-inflammatory and antifibrotic properties have been demonstrated both in vitro and in different animal models of inflammatory pathologies, including bowel and liver diseases. We review the evidence of resveratrol protective role in respiratory diseases such as acute lung injury, asthma, chronic obstructive pulmonary disease and lung fibrosis. We conclude that resveratrol and its derivatives may act as a therapeutic agents in respiratory diseases and pertinent clinical trials should be performed.

Effect of pirfenidone on proliferation, TGF-ß-induced myofibroblast differentiation and fibrogenic activity of primary human lung fibroblasts.

Pirfenidone is an orally active small molecule that has been shown to inhibit the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis. Although pirfenidone exhibits well documented antifibrotic and antiinflammatory activities, in vitro and in vivo, its molecular targets and mechanisms of action have not been elucidated. In this study, we investigated the effects of pirfenidone on proliferation, TGF-ß-induced differentiation and fibrogenic activity of primary human lung fibroblasts (HLFs). Pirfenidone reduced fibroblast proliferation and attenuated TGF-ß-induced α-smooth muscle actin (SMA) and pro-collagen (Col)-I mRNA and protein levels. Importantly, pirfenidone inhibited TGF-ß-induced phosphorylation of Smad3, p38, and Akt, key factors in the TGF-ß pathway. Together, these results demonstrate that pirfenidone modulates HLF proliferation and TGF-ß-mediated differentiation into myofibroblasts by attenuating key TGF-ß-induced signaling pathways.

Thymosin ß4 reduces IL-17-producing cells and IL-17 expression, and protects lungs from damage in bleomycin-treated mice.

Thymosin ß4 (Tß4) is a highly conserved peptide with immunomodulatory properties. In this research we investigated the effects of Tß4 on the bleomycin-induced lung damage in CD-1 mice and the changes in the number of IL-17-producing cells as well as the IL-17 expression in the lung. Male CD-1 mice were treated with bleomycin (1mg/kg) in the absence or the presence of Tß4 (6mg/kg delivered intra-peritoneally on the day of bleomycin treatment and for 2 additional doses). After sacrifice one week later, lung histology, measurement of collagen content of the lung, Broncho Alveolar Lavage Fluid (BALF) analysis, evaluation of IL17-producing cells in the blood as well as RT-PCR and IHC in the lung tissue were performed. As expected, bleomycin-induced inflammation and lung damage were substantially reduced by Tß4 treatment in CD-1 mice, as shown by the significant reduction of (i) leukocytes in BALF, (ii) histological evidence of the lung damage, and (iii) total collagen content in the lung. Importantly, the bleomycin-induced increase in the number of IL17-producing cells in the blood was significantly blocked by Tß4. Accordingly, IHC and RT-PCR results demonstrated that Tß4 substantially inhibited bleomycin-induced IL-17 over-expression in the lung tissue. This is the first report showing that a decreased amount of IL17-producing cells and inhibited IL-17 expression in the lung with Tß4 treatment correlate with its anti-inflammatory and anti-fibrotic effects.

Human lung fibroblasts increase CD4(+)CD25(+)Foxp3(+) T cells in co-cultured CD4(+) lymphocytes.

Aim of this study was to evaluate functional modifications induced by human lung fibroblasts in co-cultured CD4(+) T lymphocytes. CD4(+) T cells, resting or stimulated with ionomycin/PMA for 6h, were co-cultured with fibroblasts isolated from pulmonary biopsies, in contact or separated by a semi-permeable membrane. The expression of CD25, CTLA-4, TGF-ß, IFNγ, IL-2, IL-4, IL-10 and Foxp3 was evaluated by flow cytometric analysis. Fibroblasts induced a significant increment in CD25(+) cells in co-cultured activated CD4(+) T lymphocytes separated by a membrane. Moreover, fibroblasts treatment with a COX2 inhibitor abrogated the increment in CD25(+) cells whereas exogenous PGE2 restored it. The CD25(+) subpopulation was characterized by increased presence of Fox-P3, CTLA-4, IL-10 and TGF-ß positive cells while IFN-γ and IL-2 positive cells were diminished. Proliferative response of CD4(+) to the anti CD3/CD28-Abs was abrogated in CD4(+) co-cultured with fibroblasts thus demonstrating a suppressive feature of the expanded CD25(+) subpopulation.

Suppression of survivin induced by a BCR-ABL/JAK2/STAT3 pathway sensitizes imatinib-resistant CML cells to different cytotoxic drugs.

The BCR-ABL oncoprotein of chronic myelogenous leukemia (CML) displays exclusive cytoplasmic localization and constitutive tyrosine kinase activity leading to the activation of different pathways that favor cell proliferation and survival. BCR-ABL induces survivin expression at both the mRNA and protein level, thus inhibiting the apoptotic machinery of CML cells and contributing to the expansion of the leukemic clone. We report that, in human CML cell lines, BCR-ABL-mediated upregulation of survivin involves the JAK2/STAT3 pathway since silencing of either protein caused a consistent reduction in survivin expression. Cell lines unresponsive to imatinib mesylate (IM) because of BCR-ABL gene amplification were not resensitized to the drug after survivin downregulation. However, cells insensitive to IM because of point mutations in the BCR-ABL kinase domain were highly responsive to hydroxyurea (HU) after survivin silencing. To address the possible clinical applications of our results, we used shepherdin, a cell-permeable peptidomimetic compound that downregulates survivin expression by preventing its interaction with Hsp90. Incubation with shepherdin of immortalized cell lines both sensitive and resistant to IM enhanced cell death induced by HU and doxorubicin. Similarly, the combination of shepherdin with first- and second-generation tyrosine kinase inhibitors reduced the colony-forming potential of human progenitors derived from both patients with IM-sensitive and IM-resistant CML. These results suggest that strategies aimed at reducing survivin levels may represent a potential therapeutic option for patients with CML unresponsive to IM.

PI3K p110γ overexpression in idiopathic pulmonary fibrosis lung tissue and fibroblast cells: in vitro effects of its inhibition.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disease whose molecular pathogenesis remains unclear. In a recent paper, we demonstrated a key role for the PI3K pathway in both proliferation and differentiation into myofibroblasts of normal human lung fibroblasts treated with TGF-ß. In this research, we assessed the expression of class I PI3K p110 isoforms in IPF lung tissue as well as in tissue-derived fibroblast cell lines. Moreover, we investigated the in vitro effects of the selective inhibition of p110 isoforms on IPF fibroblast proliferation and fibrogenic activity. IHC was performed on normal and IPF lung tissue. Expression levels of PI3K p110 isoforms were evaluated by western blot and flow cytometry analysis. Fibroblast cell lines were established from both normal and IPF tissue and the effects of selective pharmacological inhibition as well as specific gene silencing by small interfering RNAs were studied in vitro. No significant differences between normal and IPF tissue/tissue-derived fibroblasts were observed for the expression of PI3K p110 α, ß and δ isoforms whereas p110γ was more greatly expressed in both IPF lung homogenates and ex vivo fibroblast cell lines. Myofibroblasts and bronchiolar basal cells in IPF lungs exhibited strong immunoreactivity for p110γ. Positive staining for the markers of proliferation proliferating cell nuclear antigen and cyclin D1 was also shown in cells of fibrolastic foci. Furthermore, both p110γ pharmacological inhibition and gene silencing were able to significantly inhibit proliferation rate as well as α-SMA expression in IPF fibroblasts. Our data suggest that PI3K p110γ isoform may have an important role in the etio-pathology of IPF and can be a specific pharmacological target.

Thymosin ß4 protects C57BL/6 mice from bleomycin-induced damage in the lung.

BACKGROUND: Thymosin ß4 (Tß4) was recently found at high concentration in the bronchoalveolar lavage fluid (BALF) of scleroderma patients with lung involvement. It has been hypothesized that Tß4 may exert a cyto-protective effect during lung injury because lower Tß4 levels were associated with interstitial lung disease progression. Moreover, Tß4 treatment prevented profibrotic gene expression in cardiac cells in vitro and in vivo. MATERIALS AND METHODS: In this study, we explored a putative Tß4 protective role in lung damage by utilizing a well-known in vivo model of lung fibrosis. C57BL/6 mice were treated with bleomycin (BLEO, 1 mg/kg) in the absence or presence of Tß4 (6 mg/kg delivered intraperitoneally on the day of BLEO treatment and for two additional doses). After sacrifice 1 week later, measurement of fluid and collagen content in the lung, BALF analysis, myeloperoxidase (MPO) activity assay, lung histology and IHC were performed. RESULTS: Compared with BLEO-treated mice, BLEO-treated mice who received Tß4 did not lose as much weight and had a higher survival rate. Moreover, BLEO-induced inflammation and lung damage were substantially reduced by Tß4 treatment, as demonstrated by the significant reduction in oedema, total collagen content, lung infiltration by leucocytes, MPO activity in lung homogenates, and histological evidence of the ongoing lung fibrosis. Results of IHC show a strong reactivity for Tß4 in the lung tissue of Tß4-treated mice. CONCLUSIONS: This is the first report that shows a Tß4 protective role in lung toxicity associated with BLEO in a mouse model. Future studies are needed to assess its putative antifibrotic properties.

Protective effects of thymosin ß4 in a mouse model of lung fibrosis.

Thymosin ß4 (Tß4) has been found to have several biological activities related to antiscarring and reduced fibrosis. For example, the anti-inflammatory properties of Tß4 and its splice variant have been shown in the eye and skin. Moreover, Tß4 treatment prevents profibrotic gene expression in cardiac and in hepatic cells in vitro and in vivo. In a recent study on scleroderma patients it was hypothesized that Tß4 may exert a protective effect during human lung injury. In an ongoing study, we have explored the putative Tß4 protective role in the lung context by utilizing a well-known in vivo model. We have observed significant protective effects of Tß4 on bleomycin-induced lung damage, the main outcomes being the halting of the inflammatory process and a substantial reduction of histological evidence of lung injury.

Inhibition of PI3K prevents the proliferation and differentiation of human lung fibroblasts into myofibroblasts: the role of class I P110 isoforms.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disease characterized by an accumulation of fibroblasts and myofibroblasts in the alveolar wall. Even though the pathogenesis of this fatal disorder remains unclear, transforming growth factor-ß (TGF-ß)-induced differentiation and proliferation of myofibroblasts is recognized as a primary event. The molecular pathways involved in TGF-ß signalling are generally Smad-dependent yet Smad-independent pathways, including phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt), have been recently proposed. In this research we established ex-vivo cultures of human lung fibroblasts and we investigated the role of the PI3K/Akt pathway in two critical stages of the fibrotic process induced by TGF-ß: fibroblast proliferation and differentiation into myofibroblasts. Here we show that the pan-inhibitor of PI3Ks LY294002 is able to abrogate the TGF-ß-induced increase in cell proliferation, in α- smooth muscle actin expression and in collagen production besides inhibiting Akt phosphorylation, thus demonstrating the centrality of the PI3K/Akt pathway in lung fibroblast proliferation and differentiation. Moreover, for the first time we show that PI3K p110δ and p110γ are functionally expressed in human lung fibroblasts, in addition to the ubiquitously expressed p110α and ß. Finally, results obtained with both selective inhibitors and gene knocking-down experiments demonstrate a major role of p110γ and p110α in both TGF-ß-induced fibroblast proliferation and differentiation. This finding suggests that specific PI3K isoforms can be pharmacological targets in IPF.

Resveratrol inhibits transforming growth factor-ß-induced proliferation and differentiation of ex vivo human lung fibroblasts into myofibroblasts through ERK/Akt inhibition and PTEN restoration.

The authors investigated the role of resveratrol (RV), a natural poliphenolic molecule with several biological activities, in transforming growth factor-ß (TGF-ß)-induced proliferation and differentiation of ex vivo human pulmonary fibroblasts into myofibroblasts. The effects of RV treatment were evaluated by analyzing TGF-ß-induced α-smooth muscle actin (α-SMA) expression and collagen production, as well as cell proliferation of both normal and idiopathic pulmonary fibrosis (IPF) lung fibroblasts. Results demonstrate that RV inhibits TGF-ß-induced cell proliferation of both normal and pathological lung fibroblasts, attenuates α-SMA expression at both the mRNA and protein levels, and also inhibits intracellular collagen deposition. In order to understand the molecular mechanisms, the authors also investigated the effects of RV treatment on signaling pathways involved in TGF-ß-induced fibrosis. The authors show that RV inhibited TGF-ß-induced phosphorylation of both extracellular signal-regulated kinases (ERK1/2) and the serine/threonine kinase, Akt. Moreover, RV treatment blocked the TGF-ß-induced decrease in phosphatase and tensin homolog (PTEN) expression levels.

Surface immobilization of fibronectin-derived PHSRN peptide on functionalized polymer films--effects on fibroblast spreading.

The Pro-His-Ser-Arg-Asn (PHSRN) sequence in fibronectin is a second cell-binding site that synergistically affects Arg-Gly-Asp (RGD). The PHSRN peptide also induces cell invasion and accelerates wound healing. We report on the surface immobilization of PHSRN by spontaneous adsorption on polysiloxane thin films which have different surface free energy characteristics. Low-surface energy (hydrophobic) polysiloxane and the corresponding high-surface energy (hydrophilic) surfaces obtained by UV-ozone treatments were used as adsorbing substrates. The peptide adsorption process was investigated by quartz crystal microbalance with dissipation monitoring and atomic force microscopy. Both adsorption kinetics and peptide rearrangement dynamics at the solid interface were significantly different on the surface-modified films compared to the untreated ones. Fibroblast cells cultures at short times and in a simplified environment, i.e., a medium-free solution, were prepared to distinguish interaction events at the interface between cell membrane and surface-immobilized peptide for the two cases. It turned out that the cell-adhesive effect of immobilized PHSRN was different for hydrophobic compared to hydrophilic ones. Early signatures of cell spreading were only observed on the hydrophilic substrates. These effects are explained in terms of different spatial arrangements of PHSRN molecules immobilized on the two types of surfaces.

16,16-Dimethyl prostaglandin E2 efficacy on prevention and protection from bleomycin-induced lung injury and fibrosis.

In this study, we evaluated the protective effect and therapeutic potential of the prostaglandin E(2) (PGE(2)) synthetic analog 16,16-dimethyl-PGE(2) (dmPGE(2)) in the animal model of pulmonary fibrosis induced by bleomycin. Mice subjected to intratracheal administration of bleomycin (1 mg/kg) received a dmPGE(2) dose of 30 microg/kg/day by continuous subcutaneous infusion. Bronchoalveolar lavage (BAL); immunohistochemical analysis for IL-1, TNF-alpha, and nitrotyrosine; measurement of fluid content in lung; myeloperoxidase activity assay; and lung histology were performed 1 week later. Lung histology and Sircol assay for collagen deposition were performed 3 weeks after treatments. Changes of body weight and survival rate were also evaluated at 1 and 3 weeks. Compared with bleomycin-treated mice, dmPGE(2) co-treated mice exhibited a reduced degree of body weight loss and mortality rate as well as of lung damage and inflammation, as shown by the significant reduction of: (1) lung infiltration by leukocytes; (2) myeloperoxidase activity; (3) IL-1, TNF-alpha, and nitrotyrosine immunostaining; (4) lung edema; and (5) histologic evidence of lung injury and collagen deposition. In a separate set of experiments, dmPGE(2) treatment was started 3 days after bleomycin administration, and the evaluation of lung damage and inflammation was assessed 4 days later. Importantly, delayed administration of dmPGE(2) also was able to protect from inflammation and lung injury induced by bleomycin. These results, indicating that dmPGE(2) is able to prevent and to reduce bleomycin-induced lung injury through its regulatory and anti-inflammatory properties, encourage further research to find new options for the treatment of pulmonary fibrosis.

Quantitative evaluation of interleukin-12 p40 gene expression in peripheral blood mononuclear cells.

The heterodimeric cytokine IL-12 (composed of a p35 and a p40 subunit) is produced primarily by monocytes, macrophages and B cells. In vitro and in vivo experiments have demonstrated the crucial role of IL-12 in initiating and establishing both innate immunity and T cell-mediated resistance to intracellular pathogens, including Leishmania donovani, Toxoplasma gondii, Listeria monocytogenes, and Mycobacterium tuberculosis. Assessment of cytokine expression has thus become crucial to understand host responses to infections. In this study, by using the reverse transcriptase-real time PCR we developed a highly specific and sensitive assay to quantitatively evaluate IL-12p40 mRNA transcription levels in peripheral blood mononuclear cells (PBMCs) stimulated with PHA vs. unstimulated cells. We also used the ELISA to evaluate bioactive IL-12 release in culture supernatants. We provide evidence that IL-12 p40 mRNA levels were significantly up-regulated in PHA-activated PBMCs. These results were correlated with data of IL-12 levels obtained by ELISA.

Peroxisomal proliferator-activated receptor-gamma agonists induce partial reversion of epithelial-mesenchymal transition in anaplastic thyroid cancer cells.

Anaplastic thyroid cancer (ATC) is an extremely aggressive tumor characterized by marked epithelial mesenchymal transition, which leads, almost invariably, to death. Peroxisomal proliferator-activated receptor (PPAR)-gamma agonists have recently emerged as potential antineoplastic drugs. To establish whether ATC could be a target of PPAR gamma agonists, we first examined PPAR gamma protein expression in a panel of six ATC cell lines and then studied the biologic effects of two PPAR gamma agonists, ciglitazone and rosiglitazone, that belong to the class of thiazolidonediones. PPAR gamma protein was present and functional in all ATC cell lines. Both ciglitazone and rosiglitazone showed complex biological effects in ATC cells, including inhibition of anchorage-dependent and -independent growth and migration, and increased apoptosis rate. Rosiglitazone-induced growth inhibition was associated with cell cycle arrest and changes in cell cycle regulators, such as an increase of cyclin-dependent kinases inhibitors p21(cip1) and p27(kip1), a decrease of cyclin D1, and inactivation of Rb protein. Rosiglitazone-induced apoptosis was associated with a decrease of Bcl-X(L) expression and caspase-3 and -7 activation. Moreover, rosiglitazone antagonized IGF-I biological effects by up-regulating phosphatase and tensin homolog deleted from chromosome 10 with subsequent inhibition of the phosphatidylinositol 3-kinase/Akt signaling pathway. Finally, rosiglitazone increased the expression of thyroid-specific differentiation markers. In conclusions, these data suggest that PPAR gamma agonists induce a partial reversion of the epithelial mesenchymal transition in ATC cells by multiple mechanisms. PPAR gamma agonists may, therefore, have a role in the multimodal therapy currently used to slow down ATC growth and dissemination.