Development of an Excipient-Free Peptide Dry Powder Inhalation for the Treatment of Pulmonary Fibrosis.

The caveolin scaffolding domain peptide (CSP) is being developed for the therapeutic intervention of a lethal lung disease, idiopathic pulmonary fibrosis. While direct respiratory delivery of CSP7 (a 7-mer fragment of CSP) is considered an effective route, proper formulation and processing of the peptide are required. First, air-jet milling technology was performed in order to micronize the neat peptide powder. Next, the fine particles were subjected to a stability study with physical and chemical characterizations. In addition, the in vivo efficacy of processed CSP7 powder was evaluated in an animal model of lung fibrosis. The results revealed that, with jet milling, the particle size of CSP7 was reduced to a mass median aerodynamic diameter of 1.58 ± 0.1 µm and 93.3 ± 3.3% fine particle fraction, optimal for deep lung delivery. A statistically significant reduction of collagen was observed in diseased lung tissues of mice that received CSP7 powder for inhalation. The particles remained chemically and physically stable after micronization and during storage. This work demonstrated that jet milling is effective in the manufacturing of a stable, excipient-free CSP7 inhalation powder for the treatment of pulmonary fibrosis.

Aerobic exercise inhibits obesity-induced respiratory phenotype.

PURPOSE: Obesity results in decreased lung function and increased inflammation. Moderate aerobic exercise (AE) reduced lung inflammation and remodeling in a variety of respiratory disease models. Therefore, this study investigated whether AE can attenuate a diet-induced obesity respiratory phenotype; including airway hyper-responsiveness (AHR), remodeling and inflammation. METHODS: Sixty C57Bl/6 male mice were distributed into four groups: control lean (CL), exercise lean (EL), obese (O) and obese exercise (OE) groups (2 sets of 7 and 8 mice per group; n = 15). A classical model of diet-induced obesity (DIO) over 12 weeks was used. AE was performed 60 min/day, 5 days/week for 5 weeks. Airway hyperresponsiveness (AHR), lung inflammation and remodeling, adipokines and cytokines in bronchoalveolar lavage (BAL) was determined. RESULTS: A high fat diet over 18 weeks significantly increased body weight (p < .0001). Five weeks of AE significantly reduced both AHR and pulmonary inflammation. AHR in obese mice that exercised was reduced at the basal level (p < .05), vehicle (PBS) (p < .05), 6.25 MCh mg/mL (p < .05), 12.5 MCh mg/mL (p < .01), 25 MCh mg/mL (p < .01) and 50 MCh mg/mL (p < .05). Collagen (p < .001) and elastic (p < .001) fiber deposition in airway wall and also smooth muscle thickness (p < .001) were reduced. The number of neutrophils (p < .001), macrophages (p < .001) and lymphocytes (p < .01) were reduced in the peribronchial space as well as in the BAL: lymphocytes (p < .01), macrophages (p < .01), neutrophils (p < .001). AE reduced obesity markers leptin (p < .001), IGF-1 (p < .01) and VEGF (p < .001), while increased adiponectin (p < .01) in BAL. AE also reduced pro-inflammatory cytokines in the BAL: IL-1ß (p < .001), IL-12p40 (p < .001), IL-13 (p < .01), IL-17 (p < .001, IL-23 (p < .05) and TNF-alpha (p < .05), and increased anti-inflammatory cytokine IL-10 (p < .05). CONCLUSIONS: Aerobic exercise reduces high fat diet-induced obese lung phenotype (AHR, pulmonary remodeling and inflammation), involving anti-inflammatory cytokine IL-10 and adiponectin.

Aerobic exercise inhibits acute lung injury: from mouse to human evidence Exercise reduced lung injury markers in mouse and in cells.

Acute respiratory distress syndrome (ARDS) is defined as hypoxemic respiratory failure with intense pulmonary inflammation, involving hyperactivation of endothelial cells and neutrophils. Given the anti-inflammatory effects of aerobic exercise (AE), this study investigated whether AE performed daily for 5 weeks would inhibit extra-pulmonary LPS-induced ARDS. C57Bl/6 mice were distributed into Control, Exercise, LPS and Exercise+LPS groups. AE was performed on a treadmill for 5x/week for four weeks before LPS administration. 24hours after the final AE physical test, animals received 100ug of LPS intra-peritoneally. In addition, whole blood cell culture, neutrophils and human endothelial cells were preincubated with IL-10, an anti-inflammatory cytokine induced by exercise. AE reduced total protein levels (p<0.01) and neutrophil accumulation in bronchoalveolar lavage (BAL) (p<0.01) and lung parenchyma (p<0.01). AE reduced BAL inflammatory cytokines IL-1ß, IL-6 and GM-CSF (p<0.001), CXCL1/KC, IL-17, TNF-alpha and IGF-1 (p<0.01). Systemically, AE reduced IL-1ß, IL-6 and IFN-gamma (p<0.001), CXCL1/KC (p<0.01) and TNF-alpha (p<0.05). AE increased IL-10 levels in serum (p<0.001) and BAL (p<0.001). Furthermore, AE increased superoxide dismutase SOD (p<0.01) and decreased superoxide anion accumulation in the lungs (p<0.01). Lastly, pre-incubation with IL-10 significantly reduced LPS-induced activation of whole blood cells, neutrophils and HUVECs, as observed by reduced production of IL-1ß, IL-6, IL-8 and TNF-alpha. Our data suggest that AE inhibited LPS-induced lung inflammation by attenuating inflammatory cytokines and oxidative stress markers in mice and human cell culture via enhanced IL-10 production.

Fgf10-positive cells represent a progenitor cell population during lung development and postnatally.

The lung mesenchyme consists of a widely heterogeneous population of cells that play crucial roles during development and homeostasis after birth. These cells belong to myogenic, adipogenic, chondrogenic, neuronal and other lineages. Yet, no clear hierarchy for these lineages has been established. We have previously generated a novel Fgf10(iCre) knock-in mouse line that allows lineage tracing of Fgf10-positive cells during development and postnatally. Using these mice, we hereby demonstrate the presence of two waves of Fgf10 expression during embryonic lung development: the first wave, comprising Fgf10-positive cells residing in the submesothelial mesenchyme at early pseudoglandular stage (as well as their descendants); and the second wave, comprising Fgf10-positive cells from late pseudoglandular stage (as well as their descendants). Our lineage-tracing data reveal that the first wave contributes to the formation of parabronchial and vascular smooth muscle cells as well as lipofibroblasts at later developmental stages, whereas the second wave does not give rise to smooth muscle cells but to lipofibroblasts as well as an Nkx2.1(-) E-Cad(-) Epcam(+) Pro-Spc(+) lineage that requires further in-depth analysis. During alveologenesis, Fgf10-positive cells give rise to lipofibroblasts rather than alveolar myofibroblasts, and during adult life, a subpopulation of Fgf10-expressing cells represents a pool of resident mesenchymal stromal (stem) cells (MSCs) (Cd45(-) Cd31(-) Sca-1(+)). Taken together, we show for the first time that Fgf10-expressing cells represent a pool of mesenchymal progenitors in the embryonic and postnatal lung. Our findings suggest that Fgf10-positive cells could be useful for developing stem cell-based therapies for treating interstitial lung diseases.

miR-142-3p balances proliferation and differentiation of mesenchymal cells during lung development.

The regulation of the balance between proliferation and differentiation in the mesenchymal compartment of the lung is largely uncharacterized, unlike its epithelial counterpart. In this study, we determined that miR-142-3p contributes to the proper proliferation of mesenchymal progenitors by controlling the level of WNT signaling. miR-142-3p can physically bind to adenomatous polyposis coli mRNA, functioning to regulate its expression level. In miR-142-3p loss-of-function experiments, proliferation of parabronchial smooth muscle cell progenitors is significantly impaired, leading to premature differentiation. Activation of WNT signaling in the mesenchyme, or Apc loss of function, can both rescue miR-142-3p knockdown. These findings show that in the embryonic lung mesenchyme, the microRNA machinery modulates the level of WNT signaling, adding an extra layer of control in the feedback loop between FGFR2C and ß-catenin-mediated WNT signaling.

Attenuating endogenous Fgfr2b ligands during bleomycin-induced lung fibrosis does not compromise murine lung repair.

Fibroblast growth factors (Fgfs) mediate organ repair. Lung epithelial cell overexpression of Fgf10 postbleomycin injury is both protective and therapeutic, characterized by increased survival and attenuated fibrosis. Exogenous administration of FGF7 (palifermin) also showed prophylactic survival benefits in mice. The role of endogenous Fgfr2b ligands on bleomycin-induced lung fibrosis is still elusive. This study reports the expression of endogenous Fgfr2b ligands, receptors, and signaling targets in wild-type mice following bleomycin lung injury. In addition, the impact of attenuating endogenous Fgfr2b-ligands following bleomycin-induced fibrosis was tested by using a doxycycline (dox)-based inducible, soluble, dominant-negative form of the Fgfr2b receptor. Double-transgenic (DTG) Rosa26(rtTA/+);tet(O)solFgfr2b mice were validated for the expression and activity of soluble Fgfr2b (failure to regenerate maxillary incisors, attenuated recombinant FGF7 signal in the lung). As previously reported, no defects in lung morphometry were detected in DTG (+dox) mice exposed from postnatal days (PN) 1 through PN105. Female single-transgenic (STG) and DTG mice were subjected to various levels of bleomycin injury (1.0, 2.0, and 3.0 U/kg). Fgfr2b ligands were attenuated either throughout injury (days 0-11; days 0-28) or during later stages (days 6-28 and 14-28). No significant changes in survival, weight, lung function, confluent areas of fibrosis, or hydroxyproline deposition were detected in DTG mice. These results indicate that endogenous Fgfr2b ligands do not significantly protect against bleomycin injury, nor do they expedite the resolution of bleomycin-induced lung injury in mice.

Aberrant expression and activity of histone deacetylases in sporadic idiopathic pulmonary fibrosis.

BACKGROUND: Activation and differentiation of fibroblasts into contractile protein-expressing myofibroblasts and their acquired apoptosis-resistant phenotype are critical factors towards the development of idiopathic pulmonary fibrosis (IPF), a fatal disease characterised by distorted pulmonary structure and excessive extracellular matrix (ECM) deposition. The molecular mechanisms underlying these processes in IPF remain incompletely understood. We investigated the possible implication of aberrant overexpression and activity of histone deacetylases (HDACs) in IPF. METHODS: We analysed lung tissues from patients with sporadic IPF (n=26) and non-diseased control lungs (n=16) for expression of class I and II HDACs. Primary IPF fibroblasts were treated with HDAC inhibitors (HDACi) LBH589 or valproic acid (VPA). RESULTS: Compared to control lungs, protein levels of class I (HDAC1, HDAC2, HDAC3, HDAC8) and class II HDACs (HDAC4, HDAC 5, HDAC 7, HDAC 9) were significantly elevated in IPF lungs. Using immunohistochemistry, strong induction of nearly all HDAC enzymes was observed in myofibroblasts of fibroblast foci and in abnormal bronchiolar basal cells at sites of aberrant re-epithelialisation in IPF lungs, but not in controls. Treatment of primary IPF fibroblasts with the pan-HDACi LBH589 resulted in significantly reduced expression of genes associated with ECM synthesis, proliferation and cell survival, as well as in suppression of HDAC7, and was paralleled by induction of endoplasmic reticulum stress and apoptosis. The profibrotic and apoptosis-resistant phenotype of IPF fibroblasts was also partly attenuated by the class I HDACi VPA. CONCLUSIONS: Aberrant overexpression of HDACs in basal cells of IPF lungs may contribute to the bronchiolisation process in this disease. Similarly, generation and apoptosis resistance of IPF fibroblasts are mediated by enhanced activity of HDAC enzymes. Therefore, pan-HDAC inhibition by LBH589 may present a novel therapeutic option for patients with IPF.

Increased FGF1-FGFRc expression in idiopathic pulmonary fibrosis.

BACKGROUND: Recent clinical studies show that tyrosine kinase inhibitors slow the rate of lung function decline and decrease the number of acute exacerbations in patients with Idiopathic Pulmonary Fibrosis (IPF). However, in the murine bleomycin model of fibrosis, not all tyrosine kinase signaling is detrimental. Exogenous ligands Fibroblast Growth Factor (FGF) 7 and 10 improve murine lung repair and increase survival after injury via tyrosine kinase FGF receptor 2b-signaling. Therefore, the level and location of FGF/FGFR expression as well as the exogenous effect of the most highly expressed FGFR2b ligand, FGF1, was analyzed on human lung fibroblasts. METHODS: FGF ligand and receptor expression was evaluated in donor and IPF whole lung homogenates using western blotting and qPCR. Immunohistochemistry for FGF1 and FGFR1/2/3/4 were performed on human lung tissue. Lastly, the effects of FGF1, a potent, multi-FGFR ligand, were studied on primary cultures of IPF and non-IPF donor fibroblasts. Western blots for pro-fibrotic markers, proliferation, FACS for apoptosis, transwell assays and MetaMorph analyses on cell cultures were performed. RESULTS: Whole lung homogenate analyses revealed decreased FGFR b-isoform expression, and an increase in FGFR c-isoform expression. Of the FGFR2b-ligands, FGF1 was the most significantly increased in IPF patients; downstream targets of FGF-signaling, p-ERK1/2 and p-AKT were also increased. Immunohistochemistry revealed FGF1 co-localization within basal cell sheets, myofibroblast foci, and Surfactant protein-C positive alveolar epithelial type-II cells as well as co-localization with FGFR1, FGFR2, FGFR3, FGFR4 and myofibroblasts expressing the migratory marker Fascin. Both alone and in the presence of heparin, FGF1 led to increased MAPK-signaling in primary lung fibroblasts. While smooth muscle actin was unchanged, heparin + FGF1 decreased collagen production in IPF fibroblasts. In addition, FGF1 + heparin increased apoptosis and cell migration. The FGFR inhibitor (PD173074) attenuated these effects. CONCLUSIONS: Strong expression of FGF1/FGFRs in pathogenic regions of IPF suggest that aberrant FGF1-FGFR signaling is increased in IPF patients and may contribute to the pathogenesis of lung fibrosis by supporting fibroblast migration and increased MAPK-signaling.

Caveolin scaffolding domain (CSD) peptide LTI-2355 modulates the phagocytic and synthetic activity of lung derived myeloid cells in Idiopathic Pulmonary Fibrosis (IPF) and Post-acute sequelae of COVID-fibrosis (PASC-F).

Rationale: The role of the innate immune system in Idiopathic Pulmonary Fibrosis (IPF) remains poorly understood. However, a functional myeloid compartment is required to remove dying cells and cellular debris, and to mediate innate immune responses against pathogens. Aberrant macrophage activity has been described in patients with Post-acute sequelae of COVID fibrosis (PASC-F). Therefore, we examined the functional and synthetic properties of myeloid cells isolated from normal donor lung and lung explant tissue from both IPF and PASC-F patients and explored the effect of LTI-2355, a Caveolin Scaffolding Domain (CSD) peptide, on these cells. Methods & Results: CD45 + myeloid cells isolated from lung explant tissue from IPF and PASC-F patients exhibited an impaired capacity to clear autologous dead cells and cellular debris. Uptake of pathogen-coated bioparticles was impaired in myeloid cells from both fibrotic patient groups independent of type of pathogen highlighting a cell intrinsic functional impairment. LTI-2355 improved the phagocytic activity of both IPF and PASC-F myeloid cells, and this improvement was paired with decreased pro-inflammatory and pro-fibrotic synthetic activity. LTI-2355 was also shown to primarily target CD206-expressing IPF and PASC-F myeloid cells. Conclusions: Primary myeloid cells from IPF and PASC-F patients exhibit dysfunctional phagocytic and synthetic properties that are reversed by LTI-2355. Thus, these studies highlight an additional mechanism of action of a CSD peptide in the treatment of IPF and progressive fibrotic lung disease.

Pre-clinical proof-of-concept of anti-fibrotic activity of caveolin-1 scaffolding domain peptide LTI-03 in ex vivo precision cut lung slices from patients with Idiopathic Pulmonary Fibrosis.

Rationale: While rodent lung fibrosis models are routinely used to evaluate novel antifibrotics, these models have largely failed to predict clinical efficacy of novel drug candidates for Idiopathic Pulmonary Fibrosis (IPF). Moreover, single target therapeutic strategies for IPF have failed and current multi-target standard of care drugs are not curative. Caveolin-1 (CAV-1) is an integral membrane protein, which, via its caveolin scaffolding domain (CSD), interacts with caveolin binding domains (CBD). CAV-1 regulates homeostasis, and its expression is decreased in IPF lungs. LTI-03 is a seven amino acid peptide derived from the CSD and formulated for dry powder inhalation; it was well tolerated in normal volunteers ( NCT04233814 ) and a safety trial is underway in IPF patients ( NCT05954988 ). Objectives: Anti-fibrotic efficacy of LTI-03 and other CSD peptides has been observed in IPF lung monocultures, and rodent pulmonary, dermal, and heart fibrosis models. This study aimed to characterize progressive fibrotic activity in IPF PCLS explants and to evaluate the antifibrotic effects of LTI-03 and nintedanib in this model. Methods: First, CBD regions were identified in IPF signaling proteins using in silico analysis. Then, IPF PCLS (n=8) were characterized by COL1A1 immunostaining, multiplex immunoassays, and bulk RNA sequencing following treatment every 12hrs with LTI-03 at 0.5, 3.0, or 10 µM; nintedanib at 0.1 µM or 1 µM; or control peptide (CP) at 10 µM. Measurements and Main Results: CBDs were present in proteins implicated in IPF, including VEGFR, FGFR and PDGFR. Increased expression of profibrotic mediators indicated active fibrotic activity in IPF PCLS over five days. LTI-03 dose dependently decreased COL1A1 staining, and like nintedanib, decreased profibrotic proteins and transcripts. Unlike nintedanib, LTI-03 did not induce cellular necrosis signals. Conclusion: IPF PCLS explants demonstrate molecular activity indicative of fibrosis during 5 days in culture and LTI-03 broadly attenuated pro-fibrotic proteins and pathways, further supporting the potential therapeutic effectiveness of LTI-03 for IPF.

Signaling by FGFR2b controls the regenerative capacity of adult mouse incisors.

Rodent incisors regenerate throughout the lifetime of the animal owing to the presence of epithelial and mesenchymal stem cells in the proximal region of the tooth. Enamel, the hardest component of the tooth, is continuously deposited by stem cell-derived ameloblasts exclusively on the labial, or outer, surface of the tooth. The epithelial stem cells that are the ameloblast progenitors reside in structures called cervical loops at the base of the incisors. Previous studies have suggested that FGF10, acting mainly through fibroblast growth factor receptor 2b (FGFR2b), is crucial for development of the epithelial stem cell population in mouse incisors. To explore the role of FGFR2b signaling during development and adult life, we used an rtTA transactivator/tetracycline promoter approach that allows inducible and reversible attenuation of FGFR2b signaling. Downregulation of FGFR2b signaling during embryonic stages led to abnormal development of the labial cervical loop and of the inner enamel epithelial layer. In addition, postnatal attenuation of signaling resulted in impaired incisor growth, characterized by failure of enamel formation and degradation of the incisors. At a cellular level, these changes were accompanied by decreased proliferation of the transit-amplifying cells that are progenitors of the ameloblasts. Upon release of the signaling blockade, the incisors resumed growth and reformed an enamel layer, demonstrating that survival of the stem cells was not compromised by transient postnatal attenuation of FGFR2b signaling. Taken together, our results demonstrate that FGFR2b signaling regulates both the establishment of the incisor stem cell niches in the embryo and the regenerative capacity of incisors in the adult.

Twisted gastrulation limits apoptosis in the distal region of the mandibular arch in mice.

The mandibular arch (BA1) is critical for craniofacial development. The distal region of BA1, which gives rise to most of the mandible, is dependent upon an optimal level of bone morphogenetic protein (BMP) signaling. BMP activity is modulated in the extracellular space by BMP-binding proteins such as Twisted gastrulation (TWSG1). Twsg1(-/-) mice have a spectrum of craniofacial phenotypes, including mandibular defects that range from micrognathia to agnathia. At E9.5, the distal region of the mutant BA1 was prematurely and variably fused with loss of distal markers eHand and Msx1. Expression of proximal markers Fgf8 and Barx1 was expanded across the fused BA1. The expression of Bmp4 and Msx2 was preserved in the distal region, but shifted ventrally. While wild type embryos showed a gradient of BMP signaling with higher activity in the distal region of BA1, this gradient was disrupted and shifted ventrally in the mutants. Thus, loss of TWSG1 results in disruption of the BMP4 gradient at the level of signaling activity as well as mRNA expression. Altered distribution of BMP signaling leads to a shift in gene expression and increase in apoptosis. The extent of apoptosis may account for the variable degree of mandibular defects in Twsg1 mutants.

The ageing lung under stress.

Healthy ageing of the lung involves structural changes but also numerous cell-intrinsic and cell-extrinsic alterations. Among them are the age-related decline in central cellular quality control mechanisms such as redox and protein homeostasis. In this review, we would like to provide a conceptual framework of how impaired stress responses in the ageing lung, as exemplified by dysfunctional redox and protein homeostasis, may contribute to onset and progression of COPD and idiopathic pulmonary fibrosis (IPF). We propose that age-related imbalanced redox and protein homeostasis acts, amongst others (e.g. cellular senescence), as a "first hit" that challenges the adaptive stress-response pathways of the cell, increases the level of oxidative stress and renders the lung susceptible to subsequent injury and disease. In both COPD and IPF, additional environmental insults such as smoking, air pollution and/or infections then serve as "second hits" which contribute to persistently elevated oxidative stress that overwhelms the already weakened adaptive defence and repair pathways in the elderly towards non-adaptive, irremediable stress thereby promoting development and progression of respiratory diseases. COPD and IPF are thus distinct horns of the same devil, "lung ageing".

Caveolin-1-derived peptide limits development of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease with a median 5-year survival of ~20%. Current U.S. Food and Drug Administration-approved pharmacotherapies slow progression of IPF, providing hope that even more effective treatments can be developed. Alveolar epithelial progenitor type II cell (AEC) apoptosis and proliferation, and accumulation of activated myofibroblasts or fibrotic lung fibroblasts (fLfs) contribute to the progression of IPF. Full-length caveolin-1 scaffolding domain peptide (CSP; amino acids 82 to 101 of Cav1: DGIWKASFTTFTVTKYWFYR) inhibits AEC apoptosis and fLf activation and expansion and attenuates PF in bleomycin (BLM)-induced lung injury in mice. Like full-length CSP, a seven-amino acid deletion fragment of CSP, CSP7 (FTTFTVT), demonstrated antifibrotic effects in murine models of lung fibrosis. When CSP7 was administered during the fibrotic phase in three preclinical models [single-dose BLM, repeated-dose BLM, and adenovirus expressing constitutively active transforming growth factor-ß1 (Ad-TGF-ß1)-induced established PF], CSP7 reduced extracellular matrix (ECM) markers characteristic of PF, increased AEC survival, and improved lung function. CSP7 is amenable to both systemic (intraperitoneal) or direct lung delivery in a nebulized or dry powder form. Furthermore, CSP7 treatment of end-stage human IPF lung tissue explants attenuated ECM production and promoted AEC survival. Ames testing for mutagenicity and in vitro human peripheral blood lymphocyte and in vivo mouse micronucleus transformation assays indicated that CSP7 is not carcinogenic. Together, these findings support the further development of CSP7 as an antifibrotic treatment for patients with IPF or other interstitial lung diseases.

Montelukast, Leukotriene Inhibitor, Reduces LPS-Induced Acute Lung Inflammation and Human Neutrophil Activation.

OBJECTIVES: Some pro-inflammatory lipids derived from 1 lipooxygenase enzyme are potent neutrophil chemoattractant, a cell centrally involved in acute respiratory distress syndrome (ARDS); a syndrome lacking effective treatment. Considering the beneficial effects of the leukotriene receptor inhibitor, montelukast, on other lung diseases, whether montelukast attenuates inflammation in a mouse model of ARDS, and whether it reduces LPS stimulated activation of human neutrophils was investigated. METHODS: Thirty-five C57Bl/6 mice were distributed into control (PBS)+24h, LPS+24h (10µg/mouse), control+48h, LPS+48h, and LPS 48h+Montelukast (10mg/kg). In addition, human neutrophils were incubated with LPS (1µg/mL) and treated with montelukast (10µM). RESULTS: Oral-tracheal administration of montelukast significantly attenuated total cells (P<.05), macrophages (P<.05), neutrophils (P<.01), lymphocytes (P<.001) and total protein levels in BAL (P<.05), as well as IL-6 (P<.05), CXCL1/KC (P<.05), IL-17 (P<.05) and TNF-α (P<.05). Furthermore, montelukast reduced neutrophils (P<.001), lymphocytes (P<.01) and macrophages (P<.01) in the lung parenchyma. In addition, montelukast restored BAL VEGF levels (P<.05). LTB4 receptor expression (P<.001) as well as NF-κB (P<.001), a downstream target of LPS, were also reduced in lung parenchymal leukocytes. Furthermore, montelukast reduced IL-8 (P<.001) production by LPS-treated human neutrophils. CONCLUSION: In conclusion, montelukast efficiently attenuated both LPS-induced lung inflammation in a mouse model of ARDS and in LPS challenged human neutrophils.

Comparison of the antifibrotic effects of the pan-histone deacetylase-inhibitor panobinostat versus the IPF-drug pirfenidone in fibroblasts from patients with idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with a poor prognosis. Pirfenidone is the first antifibrotic agent to be approved for IPF-treatment as it is able to slow down disease progression. However, there is no curative treatment other than lung transplantation. Because epigenetic alterations are associated with IPF, histone deacetylase (HDAC)-inhibitors have recently been proven to attenuate fibrotic remodeling in vitro and in vivo. This study compared the effects of pirfenidone with the pan-HDAC-inhibitor panobinostat/LBH589, a FDA-approved drug for the treatment of multiple myeloma, head-to-head on survival, fibrotic activity and proliferation of primary IPF-fibroblasts in vitro. METHODS: Primary fibroblasts from six IPF-patients were incubated for 24h with vehicle (0.25% DMSO), panobinostat (LBH589, 85 nM) or pirfenidone (2.7 mM), followed by assessment of proliferation and expression analyses for profibrotic and anti-apoptosis genes, as well as for ER stress and apoptosis-markers. In addition, the expression status of all HDAC enzymes was examined. RESULTS: Treatment of IPF-fibroblasts with panobinostat or pirfenidone resulted in a downregulated expression of various extracellular matrix (ECM)-associated genes, as compared to vehicle-treated cells. In agreement, both drugs decreased protein level of phosphorylated (p)-STAT3, a transcription factor mediating profibrotic responses, in treated IPF-fibroblasts. Further, an increase in histone acetylation was observed in response to both treatments, but was much more pronounced and excessive in panobinostat-treated IPF-fibroblasts. Panobinostat, but not pirfenidone, led to a significant suppression of proliferation in IPF-fibroblasts, as indicated by WST1- and BrdU assay and markedly diminished levels of cyclin-D1 and p-histone H3. Furthermore, panobinostat-treatment enhanced α-tubulin-acetylation, decreased the expression of survival-related genes Bcl-XL and BIRC5/survivin, and was associated with induction of ER stress and apoptosis in IPF-fibroblasts. In contrast, pirfenidone-treatment maintained Bcl-XL expression, and was neither associated with ER stress-induction nor any apoptotic signaling. Pirfenidone also led to increased expression of HDAC6 and sirtuin-2, and enhanced α-tubulin-deacetylation. But in line with its ability to increase histone acetylation, pirfenidone reduced the expression of HDAC enzymes HDAC1, -2 and -9. CONCLUSIONS: We conclude that, beside other antifibrotic mechanisms, pirfenidone reduces profibrotic signaling also through STAT3 inactivation and weak epigenetic alterations in IPF-fibroblasts, and permits survival of (altered) fibroblasts. The pan-HDAC-inhibitor panobinostat reduces profibrotic phenotypes while inducing cell cycle arrest and apoptosis in IPF-fibroblasts, thus indicating more efficiency than pirfenidone in inactivating IPF-fibroblasts. We therefore believe that HDAC-inhibitors such as panobinostat can present a novel therapeutic strategy for IPF.

Aerobic Exercise Protects from Pseudomonas aeruginosa-Induced Pneumonia in Elderly Mice.

BACKGROUND: Pseudomonas aeruginosa (PS) infection results in severe morbidity and mortality, especially in immune-deficient populations. Aerobic exercise (AE) modulates the immune system, but its effects on the outcomes of pulmonary PS infection in elderly mice are unknown. METHODS: BALB/c mice (24 weeks old) were randomized to sedentary, exercise (EX), PS, and PS + EX groups for the acute experimental setting, and PS and PS + EX groups for the chronic setting. Low-intensity AE was performed for 5 weeks, 60 min/day; 24 h after the final AE session, mice were inoculated with 5 × 104 colony-forming units (CFU) of PS, and 24 h and 14 days after PS inoculation, mice were studied. RESULTS: AE inhibited PS colonization (p < 0.001) and lung inflammation (total cells, neutrophils, lymphocytes [p < 0.01] in bronchoalveolar lavage [BAL]), with significant differences in BAL levels of IL-1ß (p < 0.001), IL-6 (p < 0.01), CXCL1 (p < 0.001), and TNF-α (p < 0.001), as well as parenchymal neutrophils (p < 0.001). AE increased BAL levels of IL-10 and parenchymal (p < 0.001) and epithelial (p < 0.001) IL-10 expression, while epithelial (p < 0.001) and parenchymal (p < 0.001) NF-κB expression was decreased. AE diminished pulmonary lipid peroxidation (p < 0.001) and increased glutathione peroxidase (p < 0.01). Pre-incubation of BEAS-2B with IL-10 inhibited PS-induced epithelial cell expression of TNF-α (p < 0.05), CD40 (p < 0.01), and dichlorodihydrofluorescein diacetate (p < 0.05). CONCLUSIONS: AE inhibits PS-induced lung inflammation and bacterial colonization in elderly mice, involving IL-10/NF-κB, and redox signaling.

Two-Way Conversion between Lipogenic and Myogenic Fibroblastic Phenotypes Marks the Progression and Resolution of Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a form of progressive interstitial lung disease with unknown etiology. Due to a lack of effective treatment, IPF is associated with a high mortality rate. The hallmark feature of this disease is the accumulation of activated myofibroblasts that excessively deposit extracellular matrix proteins, thus compromising lung architecture and function and hindering gas exchange. Here we investigated the origin of activated myofibroblasts and the molecular mechanisms governing fibrosis formation and resolution. Genetic engineering in mice enables the time-controlled labeling and monitoring of lipogenic or myogenic populations of lung fibroblasts during fibrosis formation and resolution. Our data demonstrate a lipogenic-to-myogenic switch in fibroblastic phenotype during fibrosis formation. Conversely, we observed a myogenic-to-lipogenic switch during fibrosis resolution. Analysis of human lung tissues and primary human lung fibroblasts indicates that this fate switching is involved in IPF pathogenesis, opening potential therapeutic avenues to treat patients.

Dendritic Cells Are Involved in the Effects of Exercise in a Model of Asthma.

INTRODUCTION: This study investigated the effects of aerobic exercise (AE) on both the maturation of dendritic cells (DC) and the activation of lymphocytes in a mouse model of chronic allergic airway inflammation. METHODS: C57BL/6 mice distributed into control, exercise, ovalbumin (OVA), and OVA + exercise groups were submitted to OVA sensitization and challenge. Treadmill training was performed for 4 wk, and mice were assessed for classical features of chronic allergic airway inflammation as well as dendritic cell activation and T-lymphocyte response. RESULTS: AE reduced OVA-induced eosinophilic inflammation as observed in bronchoalveolar lavage fluid (P < 0.001), airway walls (P < 0001), and also reduced collagen deposition (P < 0.001). AE also reduced bronchoalveolar lavage fluid cytokines (interleukin [IL]-4, P < 0.001; IL-5, P < 0.01; IL-6, P < 0.001; IL-13, P < 0.01; and tumor necrosis factor α, P < 0.01). Cells derived from mediastinal lymphnodes of AE animals that were restimulated with OVA produced less IL-4 (P < 0.01), IL-5 (P < 0.01), and IL-13 (P < 0.001). In addition, AE reduced both DC activation, as demonstrated by reduced release of IL-6 (P < 0.001), CXCL1/KC (P < 0.01), IL-12p70 (P < 0.01), and tumor necrosis factor α (P < 0.05) and DC maturation, as demonstrated by lower MCH-II expression (P < 0.001). CONCLUSION: AE attenuated dendritic cell and lymphocyte activation and maturation, which contributed to reduced airway inflammation and remodeling in the OVA model of chronic allergic airway inflammation.