Targeting IL-11 to reduce fibrocyte circulation and lung accumulation in animal models of pulmonary hypertension-associated lung fibrosis.

BACKGROUND AND PURPOSE: IL-11 is a member of the IL-6 family of cytokine initially considered as haematopoietic and cytoprotective factor. Recent evidence indicates that IL-11 promotes lung fibrosis and pulmonary hypertension in animal models and is elevated in lung tissue of patients with pulmonary fibrosis and pulmonary hypertension. Fibrocytes are bone marrow-derived circulating cells that participate in lung fibrosis and pulmonary hypertension, but the role of IL-11 on fibrocytes is unknown. We investigated the role of IL-11 system on fibrocyte activation in different in vitro and in vivo models of lung fibrosis associated with pulmonary hypertension. EXPERIMENTAL APPROACH: Human fibrocytes were isolated from peripheral blood of six healthy donors. Recombinant human (rh)-IL-11 and soluble rh-IL-11 receptor, α subunit (IL-11Rα) were used to stimulated fibrocytes in vitro to measure:- cell migration in a chemotactic migration chamber, fibrocyte to endothelial cell adhesion in a microscope-flow chamber and fibrocyte to myofibroblast transition. Mouse lung fibrosis and pulmonary hypertension was induced using either IL-11 (s.c.) or bleomycin (intra-tracheal), while in the rat monocrotaline (intra-tracheal) was used. In vivo siRNA-IL-11 was administered to suppress IL-11 in vivo. KEY RESULTS: RhIL-11 and soluble rhIL-11Rα promote fibrocyte migration, endothelial cell adhesion and myofibroblast transition. Subcutaneous (s.c.) IL-11 infusion elevates blood, bronchoalveolar and lung tissue fibrocytes. SiRNA-IL-11 transfection in bleomycin and monocrotaline animal models reduces blood and lung tissue fibrocytes and reduces serum CXCL12 and CXCL12/CXCR4 lung expression. CONCLUSION AND IMPLICATIONS: Targeting IL-11 reduces fibrocyte circulation and lung accumulation in animal models of pulmonary hypertension-associated lung fibrosis.

Targeting IL-11 system as a treatment of pulmonary arterial hypertension.

IL-11 is linked to fibrotic diseases, but its role in pulmonary hypertension is unclear. We examined IL-11's involvement in idiopathic pulmonary arterial hypertension (iPAH). Using samples from control (n = 20) and iPAH (n = 6) subjects, we assessed IL-11 and IL-11Rα expression and localization through RT-qPCR, ELISA, immunohistochemistry, and immunofluorescence. A monocrotaline-induced PAH model helped evaluate the impact of siRNA-IL-11 on pulmonary artery remodeling and PH. The effects of recombinant human IL-11 and IL-11Rα on human pulmonary artery smooth muscle cell (HPASMC) proliferation, pulmonary artery endothelial cell (HPAEC) mesenchymal transition, monocyte interactions, endothelial tube formation, and precision cut lung slice (PCLS) pulmonary artery remodeling and contraction were evaluated. IL-11 and IL-11Rα were over-expressed in pulmonary arteries (3.2-fold and 75-fold respectively) and serum (1.5-fold and 2-fold respectively) of patients with iPAH. Therapeutic transient transfection with siRNA targeting IL-11 resulted in a significant reduction in pulmonary artery remodeling (by 98%), right heart hypertrophy (by 66%), and pulmonary hypertension (by 58%) in rats exposed to monocrotaline treatment. rhIL-11 and soluble rhIL-11Rα induce HPASMC proliferation and HPAEC to monocyte interactions, mesenchymal transition, and tube formation. Neutralizing monoclonal IL-11 and IL-11Rα antibodies inhibited TGFß1 and EDN-1 induced HPAEC to mesenchymal transition and HPASMC proliferation. In 3D PCLS, rhIL-11 and soluble rhIL-11Rα do not promote pulmonary artery contraction but sensitize PCLS pulmonary artery contraction induced by EDN-1. In summary, IL-11 and IL-11Rα are more highly expressed in the pulmonary arteries of iPAH patients and contribute to pulmonary artery remodeling and the development of PH.

IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis.

BACKGROUND: Pulmonary hypertension (PH) associated to idiopathic pulmonary fibrosis (IPF) portends a poor prognosis. IL-11 has been implicated in fibrotic diseases, but their role on pulmonary vessels is unknown. Here we analyzed the contribution of IL-11 to PH in patients with IPF and the potential mechanism implicated. METHODS: Pulmonary arteries, lung tissue and serum of control subjects (n = 20), IPF (n = 20) and PH associated to IPF (n = 20) were used to study the expression and localization of IL-11 and IL-11Rα. Two models of IL-11 and bleomycin-induced lung fibrosis associated to PH were used in Tie2-GFP transgenic mice to evaluate the contribution of IL-11 and endothelial cells to pulmonary artery remodeling. The effect of IL-11 and soluble IL-11Rα on human pulmonary artery endothelial cells and smooth muscle cell transformations and proliferation were analyzed. RESULTS: IL-11 and IL-11Rα were over-expressed in pulmonary arteries and serum of patients with PH associated to IPF vs IPF patients without PH. Recombinant mice (rm)IL-11 induced lung fibrosis and PH in Tie2-GFP mice, activating in vivo EnMT as a contributor of pulmonary artery remodeling and lung fibrosis. Transient transfection of siRNA-IL-11 reduced lung fibrosis and PH in Tie2-GFP bleomycin model. Human (h)rIL-11 and soluble hrIL-11Rα induced endothelial to mesenchymal transition (EnMT) and pulmonary artery smooth muscle cell to myofibroblast-like transformation, cell proliferation and senescence in vitro. CONCLUSIONS: IL-11 and IL-11Rα are overexpressed in pulmonary arteries of PH associated to IPF patients, and contributes to pulmonary artery remodeling and PH.

Intake of Vitamin D in Patients with Multiple Sclerosis in the Valencian Region and Its Possible Relationship with the Pathogenesis of the Disease.

(1) Background: Multiple sclerosis (MS) is a neurodegenerative disease characterized by pronounced inflammation. Interleukin 6 (IL-6) is an accurate marker for the state of inflammation, due to the high levels of this cytokine linked to the pathogenesis of the disease. These IL-6 levels could be lowered with an adequate dietary intake of vitamin D. The objective of the study was to determine the level of vitamin D ingested in a sample of patients with MS in the Valencian region (Spain), to establish the vitamin sources, and the possible link between the intake of vitamin D and the pathogenesis of the disease through a relationship with the level of IL-6. (2) Materials and Methods: A descriptive pilot study was carried out with 39 patients with MS in the Valencian region. The dietary-nutritional anamnesis was gained through the food frequency questionnaire (FFQ) and a food diary. Diet and eating habits were analyzed through the Easy Diet (version: 2.0.1)-Consultation Management Program® software, and IL-6 levels in blood by ELISA technique. (3) Results: The results show a low intake of vitamin D, which is significantly and negatively related to the intake of proteins of vegetable origin, which are consumed in less quantity than proteins of animal origin, and significantly and negatively related with the high blood levels of IL-6, possibly as a consequence of the high intake of fats, mainly unsaturated. (4) Conclusions: MS patients in the Valencian region ingest little vitamin D related to low intake of vegetable protein, which would explain the high levels of IL-6 linked to the high intake of mainly saturated fats.

Predictive Value of Immune Cell Functional Assay for Non-Cytomegalovirus Infection in Lung Transplant Recipients: A Multicenter Prospective Observational Study.

INTRODUCTION: Immune cell functional assay (ImmuKnow®) is a non-invasive method that measures the state of cellular immunity in immunosuppressed patients. We studied the prognostic value of the assay for predicting non-cytomegalovirus (CMV) infections in lung transplant recipients. METHODS: A multicenter prospective observational study of 92 patients followed up from 6 to 12 months after transplantation was performed. Immune cell functional assay was carried out at 6, 8, 10, and 12 months. RESULTS: Twenty-three patients (25%) developed 29 non-CMV infections between 6 and 12 months post-transplant. At 6 months, the immune response was moderate (ATP 225-525ng/mL) in 14 (15.2%) patients and low (ATP<225ng/mL) in 78 (84.8%); no patients had a strong response (ATP≥525ng/mL). Only 1 of 14 (7.1%) patients with a moderate response developed non-CMV infection in the following 6 months compared with 22 of 78 (28.2%) patients with low response, indicating sensitivity of 95.7%, specificity of 18.8%, positive predictive value (PPV) of 28.2%, and negative predictive value (NPV) of 92.9% (AUC 0.64; p=0.043). Similar acute rejection rates were recorded in patients with mean ATP≥225 vs. <225ng/mL during the study period (7.1% vs. 9.1%, p=0.81). CONCLUSION: Although ImmuKnow® does not seem useful to predict non-CMV infection, it could identify patients with a very low risk and help us define a target for an optimal immunosuppression.

Predictive Value of Immune Cell Functional Assay for Non-Cytomegalovirus Infection in Lung Transplant Recipients: A Multicenter Prospective Observational Study.

INTRODUCTION: Immune cell functional assay (ImmuKnow®) is a non-invasive method that measures the state of cellular immunity in immunosuppressed patients. We studied the prognostic value of the assay for predicting non-cytomegalovirus (CMV) infections in lung transplant recipients. METHODS: A multicenter prospective observational study of 92 patients followed up from 6 to 12 months after transplantation was performed. Immune cell functional assay was carried out at 6, 8, 10, and 12 months. RESULTS: Twenty-three patients (25%) developed 29 non-CMV infections between 6 and 12 months post-transplant. At 6 months, the immune response was moderate (ATP 225-525ng/mL) in 14 (15.2%) patients and low (ATP<225ng/mL) in 78 (84.8%); no patients had a strong response (ATP≥525ng/mL). Only 1 of 14 (7.1%) patients with a moderate response developed non-CMV infection in the following 6 months compared with 22 of 78 (28.2%) patients with low response, indicating sensitivity of 95.7%, specificity of 18.8%, positive predictive value (PPV) of 28.2%, and negative predictive value (NPV) of 92.9% (AUC 0.64; p=0.043). Similar acute rejection rates were recorded in patients with mean ATP≥225 vs. <225ng/mL during the study period (7.1% vs. 9.1%, p=0.81). CONCLUSION: Although ImmuKnow® does not seem useful to predict non-CMV infection, it could identify patients with a very low risk and help us define a target for an optimal immunosuppression.

MUC4 is overexpressed in idiopathic pulmonary fibrosis and collaborates with transforming growth factor ß inducing fibrotic responses.

Several mucins are implicated in idiopathic pulmonary fibrosis (IPF); however, there is no evidence regarding the role of MUC4 in the development of IPF. Here we demonstrated that MUC4 was overexpressed in IPF patients (n = 22) compared with healthy subjects (n = 21) and located in pulmonary arteries, bronchial epithelial cells, fibroblasts, and hyperplastic alveolar type II cells. Decreased expression of MUC4 using siRNA-MUC4 inhibited the mesenchymal/myofibroblast transformations of alveolar type II A549 cells and lung fibroblasts, as well as cell senescence and fibroblast proliferation induced by TGF-ß1. The induction of the overexpression of MUC4 increased the effects of TGF-ß1 on mesenchymal/myofibroblast transformations and cell senescence. MUC4 overexpression and siRNA-MUC4 gene silencing increased or decreased, respectively, the phosphorylation of TGFßRI and SMAD3, contributing to smad-binding element activation. Immunoprecipitation analysis and confocal immunofluorescence showed the formation of a protein complex between MUC4ß/p-TGFßRI and p-SMAD3 in the cell membrane after TGF-ß1 stimulation and in lung tissue from IPF patients. Bleomycin-induced lung fibrosis was reduced in mice transiently transfected with siRNA-MUC4. This study shows that MUC4 expression is enhanced in IPF and promotes fibrotic processes in collaboration with TGF-ß1 canonical pathway that could be an attractive druggable target for human IPF.

MUC1 intracellular bioactivation mediates lung fibrosis.

BACKGROUND: Serum KL6/mucin 1 (MUC1) has been identified as a potential biomarker in idiopathic pulmonary fibrosis (IPF), but the role of MUC1 intracellular bioactivation in IPF is unknown. OBJECTIVE: To characterise MUC1 intracellular bioactivation in IPF. METHODS AND RESULTS: The expression and phosphorylation of Thr41 and Tyr46 on the intracellular MUC1-cytoplasmic tail (CT) was increased in patients with IPF (n=22) compared with healthy subjects (n=21) and localised to fibroblasts and hyperplastic alveolar type II cells. Transforming growth factor (TGF)-ß1 phosphorylated SMAD3 and thereby increased the phosphorylation of MUC1-CT Thr41 and Tyr46 in lung fibroblasts and alveolar type II cells, activating ß-catenin to form a phospho-Smad3/MUC1-CT and MUC1-CT/ß-catenin nuclear complex. This nuclear complex promoted alveolar epithelial type II and fibroblast to myofibroblast transitions, as well as cell senescence and fibroblast proliferation. The inhibition of MUC1-CT nuclear translocation using the inhibitor, GO-201 or silencing MUC1 by siRNA, reduced myofibroblast transition, senescence and proliferation in vitro. Bleomycin-induced lung fibrosis was reduced in mice treated with GO-201 and in MUC1-knockout mice. The profibrotic lectin, galectin-3, directly activated MUC1-CT and served as a bridge between the TGF-ß receptor and the MUC1-C domain, indicating TGF-ß1-dependent and TGF-ß1-independent intracellular bioactivation of MUC1. CONCLUSIONS: MUC1 intracellular bioactivation is enhanced in IPF and promotes fibrotic processes that could represent potential druggable targets for IPF.

JAK2 mediates lung fibrosis, pulmonary vascular remodelling and hypertension in idiopathic pulmonary fibrosis: an experimental study.

BACKGROUND: Pulmonary hypertension (PH) is a common disorder in patients with idiopathic pulmonary fibrosis (IPF) and portends a poor prognosis. Recent studies using vasodilators approved for PH have failed in improving IPF mainly due to ventilation (V)/perfusion (Q) mismatching and oxygen desaturation. Janus kinase type 2 (JAK2) is a non-receptor tyrosine kinase activated by a broad spectrum of profibrotic and vasoactive mediators, but its role in PH associated to PH is unknown. OBJECTIVE: The study of JAK2 as potential target to treat PH in IPF. METHODS AND RESULTS: JAK2 expression was increased in pulmonary arteries (PAs) from IPF (n=10; 1.93-fold; P=0.0011) and IPF+PH (n=9; 2.65-fold; P<0.0001) compared with PA from control subjects (n=10). PA remodelling was evaluated in human pulmonary artery endothelial cells (HPAECs) and human pulmonary artery smooth muscle cells (HPASMCs) from patients with IPF in vitro treated with the JAK2 inhibitor JSI-124 or siRNA-JAK2 and stimulated with transforming growth factor beta. Both JSI-124 and siRNA-JAK2 inhibited the HPAEC to mesenchymal transition and the HPASMCs to myofibroblast transition and proliferation. JAK2 inhibition induced small PA relaxation in precision-cut lung slice experiments. PA relaxation was dependent of the large conductance calcium-activated potassium channel (BKCa). JAK2 inhibition activated BKCa channels and reduced intracellular Ca2+. JSI-124 1 mg/kg/day, reduced bleomycin-induced lung fibrosis, PA remodelling, right ventricular hypertrophy, PA hypertension and V/Q mismatching in rats. The animal studies followed the ARRIVE guidelines. CONCLUSIONS: JAK2 participates in PA remodelling and tension and may be an attractive target to treat IPF associated to PH.

The JAK2 pathway is activated in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is the most rapidly progressive and fatal fibrotic disorder, with no curative therapies. The signal transducer and activator of transcription 3 (STAT3) protein is activated in lung fibroblasts and alveolar type II cells (ATII), thereby contributing to lung fibrosis in IPF. Although activation of Janus kinase 2 (JAK2) has been implicated in proliferative disorders, its role in IPF is unknown. The aim of this study was to analyze JAK2 activation in IPF, and to determine whether JAK2/STAT3 inhibition is a potential therapeutic strategy for this disease. METHODS AND RESULTS: JAK2/p-JAK2 and STAT3/pSTAT3 expression was evaluated using quantitative real time-PCR, western blotting, and immunohistochemistry. Compared to human healthy lung tissue (n = 10) both proteins were upregulated in the lung tissue of IPF patients (n = 12). Stimulating primary ATII and lung fibroblasts with transforming growth factor beta 1 or interleukin (IL)-6/IL-13 activated JAK2 and STAT3, inducing epithelial to mesenchymal and fibroblast to myofibroblast transitions. Dual p-JAK2/p-STAT3 inhibition with JSI-124 or silencing of JAK2 and STAT3 genes suppressed ATII and the fibroblast to myofibroblast transition, with greater effects than the sum of those obtained using JAK2 or STAT3 inhibitors individually. Dual rather than single inhibition was also more effective for inhibiting fibroblast migration, preventing increases in fibroblast senescence and Bcl-2 expression, and ameliorating impaired autophagy. In rats administered JSI-124, a dual inhibitor of p-JAK2/p-STAT3, at a dose of 1 mg/kg/day, bleomycin-induced lung fibrosis was reduced and collagen deposition in the lung was inhibited, as were JAK2 and STAT3 activation and several markers of fibrosis, autophagy, senescence, and anti-apoptosis. CONCLUSIONS: JAK2 and STAT3 are activated in IPF, and their dual inhibition may be an attractive strategy for treating this disease.

Patient-reported symptoms and functioning as indicators of mortality in advanced cystic fibrosis: A new tool for referral and selection for lung transplantation.

BACKGROUND: Despite well-known risk factors and predictive survival models, many patients with cystic fibrosis (CF) die while on the waiting list for lung transplant. We evaluated whether specific Cystic Fibrosis Questionnaire (CFQ-R) scales provide additional benefit to conventional tools in identifying referral timing and waitlist mortality. METHODS: From January 2010 to January 2015, 152 patients (34% on the waitlist) were evaluated with the CFQ-R and standard protocol quarterly. Data were used to explore the prognostic association of health-related quality of life. RESULTS: The Physical Functioning domain (PFD) of the CFQ-R predicted mortality in advanced CF disease better than habitual parameters (p = 0.005). For patients with the same forced expiratory volume in 1 sec (FEV1), a low score categorized patients with an increased risk of death. For patients with CF and FEV1 <30% predicted and a low Physical score, mortality rate was ~35% at 2 years. The best model for probability of inclusion on the waitlist was FEV1 % (p < 0.001, hazard ratio [HR] = 0.94; 95% confidence interval [CI] [0.90, 0.97]) and Physical Functioning (p = 0.013, HR = 0.96; 95% CI [0.95, 0.99]). The best model for probability of death similarly included FEV1 % (p = 0.09, HR = 0.97; 95% CI [0.94, 1.00]) and CFQ-R Physical Functioning score (p = 0.005, HR = 0.97; 95% CI [0.95, 0.99]). The Health Perception score showed similar results. A low Health Perception score combined with a high resting heart rate showed a trend for mortality. CONCLUSIONS: The CFQ-R may be an additional tool for guiding decisions to place a patient with CF on the waiting list for lung transplantation. The CFQ-R Physical Functioning and Health Perception scales were more accurate than conventional tools in predicting death before transplant.

Vascular effects of sildenafil in patients with pulmonary fibrosis and pulmonary hypertension: an ex vivo/in vitro study.

Sildenafil improves the 6-min walking distance in patients with idiopathic pulmonary fibrosis (IPF) and right-sided ventricular systolic dysfunction.We analysed the previously unexplored role of sildenafil on vasoconstriction and remodelling of pulmonary arteries from patients with IPF and pulmonary hypertension (PH) ex vivo Pulmonary arteries from 18 donors without lung disease, nine IPF, eight PH+IPF and four PH patients were isolated to measure vasodilator and anti-contractile effects of sildenafil in isometric organ bath. Ventilation/perfusion was explored in an animal model of bleomycin lung fibrosis.Sildenafil relaxed serotonin (5-HT) pre-contracted pulmonary arteries in healthy donors and IPF patients and, to a lesser extent, in PH+IPF and PH. Sildenafil inhibited 5-HT dose-response contraction curve mainly in PH+IPF and PH, but not in healthy donors. Sildenafil did not impair the ventilation/perfusion mismatching induced by bleomycin. Pulmonary arteries from PH+IPF patients showed a marked expression of phosphodiesterse-5 and extracellular matrix components. Sildenafil inhibited pulmonary artery endothelial and smooth muscle cell to mesenchymal transition by inhibition of extracellular regulated kinases 1 and 2 (ERK1/2) and SMAD3 phosphorylation.These results suggest an absence of direct relaxant effect and a prominent anti-contractile and anti-remodelling role of sildenafil in PH+IPF pulmonary arteries that could explain the beneficial effects of sildenafil in IPF with PH phenotype.

Impact of Single Nucleotide Polymorphisms (SNPs) on Immunosuppressive Therapy in Lung Transplantation.

Lung transplant patients present important variability in immunosuppressant blood concentrations during the first months after transplantation. Pharmacogenetics could explain part of this interindividual variability. We evaluated SNPs in genes that have previously shown correlations in other kinds of solid organ transplantation, namely ABCB1 and CYP3A5 genes with tacrolimus (Tac) and ABCC2, UGT1A9 and SLCO1B1 genes with mycophenolic acid (MPA), during the first six months after lung transplantation (51 patients). The genotype was correlated to the trough blood drug concentrations corrected for dose and body weight (C0/Dc). The ABCB1 variant in rs1045642 was associated with significantly higher Tac concentration, at six months post-transplantation (CT vs. CC). In the MPA analysis, CT patients in ABCC2 rs3740066 presented significantly lower blood concentrations than CC or TT, three months after transplantation. Other tendencies, confirming previously expected results, were found associated with the rest of studied SNPs. An interesting trend was recorded for the incidence of acute rejection according to NOD2/CARD15 rs2066844 (CT: 27.9%; CC: 12.5%). Relevant SNPs related to Tac and MPA in other solid organ transplants also seem to be related to the efficacy and safety of treatment in the complex setting of lung transplantation.

[Balloon dilatation of the trachea as treatment for idiopathic tracheal stenosis]. / Dilatación traqueal neumática en el tratamiento de la estenosis traqueal idiopática.

The treatment of choice for idiopathic tracheal stenosis is tracheal resection and anastomosis, although some authors prefer more conservative management. Between January 1, 1996 and January 1, 2005, 8 patients-all women-with idiopathic tracheal stenosis were treated in the chest surgery department of the Hospital Universitario La Fe in Valencia, Spain. One case was treated by means of surgery and so was excluded from this study. The remaining 7 women were treated by tracheal balloon dilatation; 4 required just 1 dilatation (and remained asymptomatic), 2 required 2 dilatations, and 1 required 4 dilatations. The median symptom-free interval was 25.5 months, and there was no associated mortality or morbidity. We conclude that balloon dilatation, which was not associated with mortality or morbidity, is a suitable treatment option for idiopathic tracheal stenosis.

Donor fat embolism and primary graft dysfunction after lung transplantation.

Primary lung graft dysfunction is one of the major causes of perioperative morbidity and mortality in lung transplantation. Primary lung graft dysfunction is a clinical syndrome occurring in the immediate postoperative period after lung transplantation and is characterized by severe hypoxemia, pulmonary edema, and pulmonary infiltrates on chest x-ray film, requiring that the patient remain intubated and thus favoring pulmonary infection, sepsis, and subsequent multiple organ failure in the transplanted patient. It has recently been shown that unexpected pulmonary embolism is relatively common in the donor and is associated with primary lung graft dysfunction. However, we believe that only one case of primary lung graft dysfunction due to pulmonary fat embolism has been documented histologically in patients undergoing lung transplantation. The objective of this study is to report our experience with a case of primary lung graft dysfunction due to fat embolism in the donor lung detected in the morphologic study.

[Pneumonectomy in octogenarian patients]. / Neumonectomía en pacientes octogenarios.

Recent decades have witnessed a progressive aging of the population and a resulting increase in the numbers of elderly patients seeking health care. Since age and pneumonectomy are independent predictors of perioperative morbidity and mortality, such surgery is not recommended for octogenarian patients. We report the experience of 6 such patients who underwent pneumonectomy for squamous cell carcinoma in 5 cases and a typical carcinoid tumor in the sixth. There was no perioperative mortality; morbidity and survival rates were acceptable.