Melatonin Enhances Neural Differentiation of Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (ASCs) are adult multipotent stem cells, able to differentiate toward neural elements other than cells of mesodermal lineage. The aim of this research was to test ASC neural differentiation using melatonin combined with conditioned media (CM) from glial cells. Isolated from the lipoaspirate of healthy donors, ASCs were expanded in a basal growth medium before undergoing neural differentiation procedures. For this purpose, CM obtained from olfactory ensheathing cells and from Schwann cells were used. In some samples, 1 µM of melatonin was added. After 1 and 7 days of culture, cells were studied using immunocytochemistry and flow cytometry to evaluate neural marker expression (Nestin, MAP2, Synapsin I, GFAP) under different conditions. The results confirmed that a successful neural differentiation was achieved by glial CM, whereas the addition of melatonin alone did not induce appreciable changes. When melatonin was combined with CM, ASC neural differentiation was enhanced, as demonstrated by a further improvement of neuronal marker expression, whereas glial differentiation was attenuated. A dynamic modulation was also observed, testing the expression of melatonin receptors. In conclusion, our data suggest that melatonin's neurogenic differentiation ability can be usefully exploited to obtain neuronal-like differentiated ASCs for potential therapeutic strategies.

Circulating Coding and Long Non-Coding RNAs as Potential Biomarkers of Idiopathic Pulmonary Fibrosis.

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is a chronic degenerative disease with a median survival of 2-5 years after diagnosis. Therefore, IPF patient identification represents an important and challenging clinical issue. Current research is still searching for novel reliable non-invasive biomarkers. Therefore, we explored the potential use of long non-coding RNAs (lncRNAs) and mRNAs as biomarkers for IPF. METHODS: We first performed a whole transcriptome analysis using microarray (n = 14: 7 Control, 7 IPF), followed by the validation of selected transcripts through qPCRs in an independent cohort of 95 subjects (n = 95: 45 Control, 50 IPF). Diagnostic performance and transcript correlation with functional/clinical data were also analyzed. RESULTS: 1059 differentially expressed transcripts were identified. We confirmed the downregulation of FOXF1 adjacent non-coding developmental regulatory RNA (FENDRR) lncRNA, hsa_circ_0001924 circularRNA, utrophin (UTRN) and Y-box binding protein 3 (YBX3) mRNAs. The two analyzed non-coding RNAs correlated with Forced Vital Capacity (FVC)% and Diffusing Capacity of the Lung for carbon monoxide (DLCO)% functional data, while coding RNAs correlated with smock exposure. All analyzed transcripts showed excellent performance in IPF identification with Area Under the Curve values above 0.87; the most outstanding one was YBX3: AUROC 0.944, CI 95% = 0.895-0.992, sensitivity = 90%, specificity = 88.9%, p-value = 1.02 × 10-13. CONCLUSIONS: This study has identified specific transcript signatures in IPF suggesting that validated transcripts and microarray data could be useful for the potential future identification of RNA molecules as non-invasive biomarkers for IPF.

Pharmacological treatment in Idiopathic Pulmonary Fibrosis: current issues and future perspectives

Idiopathic pulmonary fibrosis (IPF) represents a fibrotic interstitial lung disease characterized by uncertain etiology and poor prognosis. Over the years, the path to effective treatments has been marked by a series of advances and setbacks. The introduction of approved antifibrotic drugs, pirfenidone and nintedanib, marked a pivotal moment in the management of IPF. However, despite these advances, these drugs are not curative, although they can slow the natural progression of the disease. The history of drug therapy for IPF goes together with the increased understanding of the pathogenic mechanisms underlying the disease. Based on that, current research efforts continue to explore new therapies, possible personalized treatment strategies, drug combinations, and potential biomarkers for diagnosis and prognosis. In this review, we outline the route that led to the discover of the first effective therapies, ongoing clinical trials, and future directions in the search for more effective treatments.

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.

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.

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.

Clinical and Prognostic Significance of p-ANCA Positivity in Idiopathic Pulmonary Fibrosis: A Retrospective Observational Study.

Perinuclear Anti Neutrophil Cytoplasmic Antibody (p-ANCA) is a serological marker of Microscopic Polyangiitis (MPA), a vasculitis associated with lung involvement potentially mimicking Idiopathic Pulmonary Fibrosis (IPF). In this study, we evaluated the role of p-ANCA in predicting clinical evolution and prognosis in a cohort of IPF patients. In this observational, retrospective, case-control study, we compared 18 patients with an IPF diagnosis and p-ANCA positivity with 36 patients with seronegative IPF, matched for age and sex. IPF patients with and without p-ANCA showed similar lung function decline during the follow-up, but IPF p-ANCA+ showed better survival. Half of IPF p-ANCA+ patients were classified as MPA for the development of renal involvement (55%) or skin signs (45%). The progression towards MPA was associated with high levels of Rheumatoid Factor (RF) at baseline. In conclusion, p-ANCA, mainly when associated with RF, could predict the evolution of Usual Interstitial Pneumonia (UIP) towards a definite vasculitis in patients, with a better prognosis compared with IPF. In this view, ANCA testing should be included in the diagnostic workup of UIP patients.

Myositis-Specific and Myositis-Associated Antibodies in Fibromyalgia Patients: A Prospective Study.

Fibromyalgia (FM) is a common rheumatologic disorder characterised by widespread muscular pain. Myalgia is also a common clinical feature in Connective Tissue Disease (CTD), and FM should be studied for the concomitant presence of a CTD. The aim of this study is to evaluate the prevalence of Myositis-Specific and Myositis-Associated Antibodies (MSA/MAA) in a cohort of FM patients. We enrolled 233 consecutive FM patients (defined according to the 2016 criteria) that did not report clinical signs of autoimmune disorders and followed them for at least one year. The patients were tested for MSA/MAA with immunoblotting. FM patients were seropositive for Antinuclear Antibodies (ANA) in 24% of cases, for MSA in 9%, and for MAA in 6%. A specific diagnosis of CTD was made in 12 patients (5.2%), namely, 5 cases of primary Sjögren's Syndrome and 7 of Idiopathic Inflammatory Myopathy. Seropositive patients showed clinical features similar to those who were seronegative at baseline. A CTD diagnosis was associated with ANA positivity (p = 0.03, X2 4.9), the presence of a speckled pattern (p = 0.02, X2 5.3), positivity for MAA (p = 0.004, X2 8.1), and MSA (p = 0.003, X2 9.2). In conclusion, a non-negligible proportion of FM patients may be seropositive for MSA/MAA, and that seropositivity might suggest a diagnosis of CTD.

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.

Reciprocal Interplay Between Astrocytes and CD4+ Cells Affects Blood-Brain Barrier and Neuronal Function in Response to ß Amyloid.

Background: In Alzheimer's disease (AD) neuronal degeneration is associated with gliosis and infiltration of peripheral blood mononuclear cells (PBMCs), which participate in neuroinflammation. Defects at the blood-brain barrier (BBB) facilitate PBMCs migration towards the central nervous system (CNS) and in particular CD4+ T cells have been found in areas severely affected in AD. However, the role of T cells, once they migrate into the CNS, is not well defined. CD4+ cells interact with astrocytes able to release several factors and cytokines that can modulate T cell polarization; similarly, astrocytic properties are modulated after interaction with T cells. Methods: In in vitro models, astrocytes were primed with ß-amyloid (Aß; 2.5 µM, 5 h) and then co-cultured with magnetically isolated CD4+ cells. Cytokines expression was evaluated both in co-cultured CD4+ cells and astrocytes. The effects of this crosstalk were further evaluated by co-culturing CD4+ cells with the neuronal-like SH-SY5Y cell line and astrocytes with endothelial cells. Results: The pattern of cytokines and trophic factors expressed by CD4+ cells were strongly modulated in the presence of Aß-primed astrocytes. Specifically, the percentage of IL-4+ and IFNγ+ CD4+ cells was significantly increased and reduced, respectively. Further, increased BDNF mRNA levels were observed in CD4+ cells. When SH-SY5Y cells were co-cultured with astrocyte-conditioned CD4+ cells and exposed to Aß, the reduction of the presynaptic protein synaptophysin was prevented with a BDNF-dependent mechanism. In astrocytes co-cultured with CD4+ cells, reduced mRNA levels of inflammatory cytokines and VEGF were observed. This was paralleled by the prevention of the reduction of claudin-5 when astrocytes were co-cultured with endothelial cells. Conclusion: Following Aß exposure, there exists reciprocal crosstalk between infiltrating peripheral cells and astrocytes that in turn affects not only endothelial function and thus BBB properties, but also neuronal behavior. Since astrocytes are the first cells that lymphocytes interact with and are among the principal players in neuroinflammation occurring in AD, understanding this crosstalk may disclose new potential targets of intervention in the treatment of neurodegeneration.

Nailfold Videocapillaroscopy is a Useful Tool to Recognize Definite Forms of Systemic Sclerosis and Idiopathic Inflammatory Myositis in Interstitial Lung Disease Patients.

Nailfold videocapillaroscopy (NVC) is an easy tool used for the assessment of patients with Raynaud's phenomenon (RP) as possibly associated with systemic sclerosis (SSc). Recent insights have also highlighted its role in the diagnostic assessment of idiopathic inflammatory myopathies (IIMs). The aim of this study is to describe the diagnostic role of NVC in a series of 361 consecutive patients with interstitial lung disease (ILD). All the patients were assessed by clinical pulmonary and rheumatic examinations, blood exams, high-resolution computed tomography and NVC. NVC was considered positive only in the presence of avascular areas or giant capillaries, but also, the presence of bushy capillaries (BCs) was recorded. NVC was positive in 17.7% of ILD patients and in 78.1% of ILD patients associated with a diagnosis of connective tissue disease (CTD). In 25% of SSc-ILD patients, NVC proved necessary for a correct diagnosis. The presence of BCs and/or NVC positivity in ILD patients with normal levels of creatine phosphokinase is associated with amyopathic IIM, regardless the presence of RP. In conclusion, NVC is useful for the diagnostic assessment of incomplete forms of CTD and in amyopathic IIMs. NVC should be considered in the diagnostic assessment of ILD patients regardless of the presence of RP.

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.

Astrocytes Modify Migration of PBMCs Induced by ß-Amyloid in a Blood-Brain Barrier in vitro Model.

BACKGROUND: The brain is protected by the blood-brain barrier (BBB), constituted by endothelial cells supported by pericytes and astrocytes. In Alzheimer's disease a dysregulation of the BBB occurs since the early phases of the disease leading to an increased access of solutes and immune cells that can participate to the central inflammatory response. Here we investigated whether astrocytes may influence endothelial-leukocytes interaction in the presence of amyloid-ß (Aß). METHODS: We used an in vitro BBB model, where endothelial cells, cultured alone or with astrocytes were exposed for 5 h to Aß, both under resting or inflammatory conditions (TNFα and IFNγ), to evaluate endothelial barrier properties, as well as transendothelial migration of peripheral blood mononuclear cells (PBMCs). RESULTS: In the co-culture model, barrier permeability to solutes was increased by all treatments, but migration was only observed in inflammatory conditions and was prevented by Aß treatment. On the contrary, in endothelial monocultures, Aß induced leukocytes migration under resting conditions and did not modify that induced by inflammatory cytokines. In endothelial astrocyte co-cultures, a low molecular weight (MW) isoform of the adhesion molecule ICAM-1, important to allow interaction with PBMCs, was increased after 5 h exposure to inflammatory cytokines, an effect that was prevented by Aß. This modulation by Aß was not observed in endothelial monocultures. In addition, endothelial expression of ß-1,4-N-acetylglucosaminyltransferase III (Gnt-III), responsible for the formation of the low MW ICAM-1 isoform, was enhanced in inflammatory conditions, but negatively modulated by Aß only in the co-culture model. miR-200b, increased in astrocytes following Aß treatment and may represent one of the factors involved in the control of Gnt-III expression. CONCLUSION: These data point out that, at least in the early phases of Aß exposure, astrocytes play a role in the modulation of leukocytes migration through the endothelial layer.

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.

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.

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.

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.

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.