Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease with limited treatment options. LTI-03 promotes alveolar epithelial cell survival and reduces profibrotic protein expression in experimental models of IPF. In this Phase 1b, randomized, double-blind, placebo-controlled dose-escalation study, 24 participants with IPF were randomized 3:1 to inhaled LTI-03 5 mg/day (N = 9), LTI-03 10 mg/day (N = 9) or placebo (N = 6) for 14 days and included in all analyses (ClinicalTrials.gov: NCT05954988). The primary endpoint was the incidence of treatment-emergent adverse events (TEAEs). Exploratory analyses included pharmacokinetics and disease-related biomarkers. LTI-03 was well-tolerated, with no treatment-related discontinuations, no severe TEAEs, and no evidence of airway obstruction by spirometry and associated symptoms. In deep bronchial brushings, both LTI-03 doses significantly reduced interleukin-11 (p = 0.0406 at 5 mg/day; p = 0.044 at 10 mg/day) and thymic stromal lymphopoietin (p = 0.0256 at 5 mg/day; p = 0.0128 at 10 mg/day) versus placebo. The 10 mg/day dose suppressed collagen type 1 alpha chain 1 (p = 0.0248), CXC chemokine ligand 7 (p = 0.0248) and galectin-7 (p = 0.0332). Other measured biomarkers were not significantly changed. The favorable safety profile and reductions in disease-related biomarkers support further evaluation of inhaled LTI-03 for IPF. This study was fully funded by Rein Therapeutics, Inc.

Idiopathic pulmonary fibrosis (IPF) features excessive extracellular matrix deposition driven by activated fibroblasts and dysregulated signaling pathways. To assess the anti-fibrotic effects of LTI-03, a caveolin-1 scaffolding domain peptide, ex vivo precision-cut lung slices (PCLS) from patients with IPF were evaluated using bulk RNA sequencing, Ingenuity Pathway Analysis, and immunofluorescence. LTI-03 dose-dependently reduced collagen protein levels, suppressed pro-fibrotic cytokines, inhibited pro-fibrotic pathways, and activated protective mechanisms such as PTEN and PPAR signaling. Modulation was comparable to nintedanib but without the induction of apoptosis or necrosis pathways. These results demonstrate LTI-03's potential therapeutic efficacy in a highly relevant translational disease model and support LTI-03 as a promising next-generation therapeutic to halt IPF progression and improve patient outcomes.

Rationale:LTI-03 is a novel inhaled therapeutic in development for idiopathic pulmonary fibrosis (IPF). LTI-03 has been shown to promote alveolar epithelial cell survival and reduce profibrotic protein expression in experimental models of lung disease. Objectives:To evaluate the safety and pharmacokinetics (PK) of LTI-03 and effects on disease biomarkers in patients with IPF. Methods:This Phase 1b, randomized, controlled, dose-escalation study randomized 24 participants, 3:1 into 2 sequential dose cohorts, to receive inhaled LTI-03 (5 or 10 mg/day) or placebo for 14 days. The primary endpoint was the incidence of treatment-related adverse events (TEAEs). Exploratory analyses included PK and change from baseline in fibrosis-related and epithelial integrity-related biomarkers in plasma, peripheral blood mononuclear cells (PBMCs), and deep bronchial brushings (DBB). Measurements and Main Results:Inhaled LTI-03 was well-tolerated, with no treatment-related AEs leading to treatment discontinuation. All TEAEs were mild or moderate in severity. Cough was the most common TEAE and the only treatment-related TEAE experienced by more than 1 participant. There was no evidence of airway obstruction by symptoms or spirometry. LTI-03 did not induce inflammation (phosphorylated AKT) in PBMCs. In DBB samples, LTI-03 significantly reduced the expression of interleukin-11, chemokine ligand 7, thymic stromal lymphopoietin, and galectin 7; dose-related reductions were also observed for collagen type 1 alpha chain 1 and plasma surfactant protein D. Conclusions:Inhaled LTI-03 exhibited a favorable safety and tolerability profile over 14 days. Exploratory biomarker analyses suggest a positive effect on epithelial homeostasis and corresponding antifibrotic effects. At A Glance:Scientific Knowledge on the Subject: Current standard of care therapies for IPF do not halt or reverse disease progression, and systemic side effects commonly result in discontinued use. LTI-03 is a caveolin scaffolding domain (CSD) peptide formulated as an excipient-free dry powder for the treatment of IPF. It has demonstrated anti-fibrotic and epithelial protective effects in animal models of fibrosis and in IPF precision cut lung slices via putative replenishment of endogenous Cav-1 signaling, which is lost in fibrotic diseases.What This Study Adds to the Field: The results of this study support the conclusion that LTI-03 was well tolerated in patients with IPF at doses of 5 and 10 mg/day. Exploratory biomarker analyses from deep bronchial brushings suggested that positive pharmacodynamic effects were achieved on target cell types. Taken together, these results support the initiation of a Phase 2 efficacy study in patients with IPF. Data sharing statement:Rein Therapeutics, Inc. ("Rein") understands and acknowledges the need to share clinical study data with the research community in an open and transparent manner and has provided de-identified patient data in the manuscript. Rein will not consider further requests pertaining to clinical data outside of what has been accepted and published by the journal. Any queries regarding clinical study data must be submitted in writing to https://info@reintx.com . A data supplement for this article is available via the Supplements tab at the top of the online article.

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, as well as to mediate innate immune responses against pathogens. Aberrant macrophage activity has been described in patients with post-acute sequelae of COVID fibrosis (PASC-F), and caveolin scaffolding domain (CSD) peptides have been found to attenuate inflammation and fibrosis in mouse lung injury models. Therefore, we examined, for the first time, the effects of CSD peptide LTI-2355 on the functional and synthetic properties of human myeloid cells isolated from lung explant tissue of donor lungs as well as IPF and PASC-F lung explant tissue. Methods and 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. The uptake of pathogen-coated bioparticles was impaired in myeloid cells from both fibrotic patient groups independent of the type of pathogen, highlighting an intrinsic functional cell impairment. LTI-2355 improved the phagocytic activity of both IPF and PASC-F myeloid cells, and this improvement was paired with decreased proinflammatory 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 modulated by LTI-2355. LTI-2355 treatment of IPF myeloid cells resulted in significantly reduced sCD163, IFN-α2, IFN-γ, IL-2, IL-10, IL-12p40, and MMP-1 in the cell supernatant. This study highlights an additional mechanism of action of the CSD peptide in the treatment of IPF and progressive fibrotic lung disease.

Rationale: Idiopathic pulmonary fibrosis is a progressive disease characterized by unrelenting lung remodeling ultimately leading to lung failure and death. Single cell RNAseq studies have revealed that there is marked decrease in the abundance of alveolar type 2 cells (AT2) in lung samples from IPF patients. Reduction of AT2 viability impairs alveolar regeneration since these cells are a source of alveolar type 1 cells (AT1). In this study, we optimized a 3D alveolosphere model generated with isolated and cultured alveolar type 2 cells from IPF and donor, non-IPF control patients and evaluated the effect of CSD peptides LTI-03 and LTI-2355.Methods: Cryo-preserved IPF and normal donor lung explants were processed using enzymatic and mechanical digestion to obtain lung epithelial cell preparations. Primary human AT2 cells (EPCAM+ HTII-280+) were isolated using fluorescence-activated cell sorting (FACS). FACS-sorted AT2 cells were subsequently resuspended in Matrigel (1:1 Matrigel to media). A 30 μL drop containing 5 x104 AT2 cells was applied to the center of each well in a 24 well plate. Either vehicle control, CSD peptide (LTI-03 or LTI-2355 at 0.5, 3, or 10 μM) or Nintedanib (80 nM) were added into each drop, and to the overlying tissue culture media. Treatments were changed every other day for 28 days. Results: Both LTI-03 and LTI-2355 CSD peptides at the 10 μM dose increased the number of IPF alveolosperes compared with the control treatment of IPF alveolospheres at day 28 in culture. CSD peptide treatment did not alter the average size of IPF alveolospheres. Quantitative PCR analysis of SFTPC levels in IPF and normal donor alveolospheres revealed that CSD peptides increase SFTPC transcripts in both groups of alvelospheres. Finally, Nintedanib treatment of normal donor alveolospheres significantly reduced the number and size of alveolospheres at day 28 of culture compared with vehicle or CSD treated normal donor alveolospheres.Conclusion: LTI-03 and LTI-2355 increased the outgrowth of IPF alveolospheres in 3D tissue culture indicating that LTI CSD peptides exert a putative pro-regenerative effect on IPF AT2 cells. In contrast, nintedanib exhibited inhibitory effects on the outgrowth of normal donor alveolospheres. Ethical approval: Institutional Review Boards both at Cedars-Sinai Medical Center and Aileron Therapeutics approved all experiments with human tissue.

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.

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.### Competing Interest StatementBreAnne MacKenzie is an employee of Lung Therapeutics, Inc. Cory Hogaboam is a consulting CSO for Lung Therapeutics, Inc.* AM : Alveolar macrophage. CAV : Caveolin CD : Cluster of differentiation CH3IL1 : Chitinase 3-like-1 COPD : Chronic obstructive pulmonary disease CSD : Caveolin scaffolding domain DMEM : Dulbecco’s Modified Eagle Medium ECM : Extracellular matrix FABP : Fatty Acid Binding Protein IL : Interleukin IPF : Idiopathic Pulmonary Fibrosis MERTK MER : Proto-Oncogene, Tyrosine Kinase MMR/MRC : Mannose receptor MMP : Matrix metalloprotease Mo-MA : Monocyte derived macrophage PASC-F : Post-acute sequelae of COVID fibrosis RCU : Red Calibrated Unit SA : Staphylococcus aureus sCD : soluble CD SPP/OPN : Osteopontin TLR : Toll like receptor TNF : Tumor necrosis factor TR-AM : Tissue-resident alveolar macrophage

CSP7, a caveolin-1–derived peptide, targets injured alveolar epithelial cells and activated lung fibroblasts to mitigate 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.

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”.

Idiopathic pulmonary fibrosis (IPF) is associated with progressive destruction of the lung parenchyma with a median survival after diagnosis of three years and a five-year survival of

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. Thirty-five C57Bl/6 mice were distributed into control (PBS) + 24 h, LPS + 24 h (10 μg/mouse), control + 48 h, LPS + 48 h, and LPS 48 h + Montelukast (10 mg/kg). In addition, human neutrophils were incubated with LPS (1 μg/mL) and treated with montelukast (10 μM). 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. In conclusion, montelukast efficiently attenuated both LPS-induced lung inflammation in a mouse model of ARDS and in LPS challenged human neutrophils. Algunos lípidos proinflamatorios derivados de la enzima lipooxigenasa 1 son potentes quimioatrayentes de neutrófilos, un tipo celular con una implicación principal en el síndrome de distrés respiratorio agudo (SDRA), para el que no hay tratamiento efectivo. Considerando los efectos beneficiosos del inhibidor de los receptores de leucotrienos montelukast en otras enfermedades pulmonares, se investigó si este fármaco era capaz de atenuar la inflamación en un modelo de ratón de SDRA y de reducir la activación de los neutrófilos humanos inducida por LPS. Se utilizaron 35 ratones C57BL/6 distribuidos en los siguientes grupos: control (PBS) + 24 h, LPS + (24 h [10 μg/ratón]), control + 48 h y LPS 48 h + montelukast (10 mg/kg). Por otro lado, se incubaron neutrófilos humanos con LPS (1 μg/ml) y se trataron con montelukast (10 μM). La administración orotraqueal de montelukast redujo el número total de células (p < 0,05), de macrófagos (p < 0,05), de neutrófilos (p < 0,01), de linfocitos (p < 0,001) y los niveles totales de proteína en el lavado broncoalveolar (p < 0,05), así como de IL-6 (p < 0,05), CXCL1/KC (p < 0,05), IL-17 (p < 0,05) y TNF-α (p < 0,05). Además, el montelukast redujo los neutrófilos (p < 0,001), los linfocitos (p < 0,01) y los macrófagos (p < 0,01) en el parénquima pulmonar. Asimismo, restauró los niveles de VEGF en el lavado broncoalveolar (p < 0,05) y disminuyó la expresión del receptor LTB4 (p < 0,001) y de NF-κB (p < 0,001), una diana downstream del LPS, en los leucocitos del parénquima pulmonar. Por último, redujo la producción de IL-8 por parte de los neutrófilos humanos tratados con LPS. En conclusión, el montelukast atenuó de manera eficaz tanto la inflamación pulmonar inducida por LPS en un modelo de ratón de SDRA como en neutrófilos humanos estimulados con LPS.

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-1β (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.

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.

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-1β, IL-6 and GM-CSF (p<0.001), CXCL1/KC, IL-17, TNF-alpha and IGF-1 (p<0.01). Systemically, AE reduced IL-1β, 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-1β, 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.

Die idiopathische pulmonale Fibrose (IPF) ist eine schwerwiegende und in der Regel zum Tod führende Erkrankung, die bisher nur unzureichend behandelt werden kann. Für die irreversible Vernarbung der Lunge und die exzessive Synthese extrazellulärer Matrix (EZM)-Proteine sind die Myofibroblasten in den sogenannten Fibroblast Foci verantwortlich, welche in der IPF in einem erheblichen Umfang entstehen und durch Apoptose-Resistenz gekennzeichnet sind. Wir konnten zudem beobachten, dass IPF-Fibroblasten eine abnormal gesteigerte Histondeacetylase (HDAC)-Aktivität aufweisen. Ziel unserer Studie war daher, die therapeutische Effizienz des pan-HDAC-Inhibitors LBH589 im Vergleich zu dem IPF-Medikament Pirfenidon in primären Lungenfibroblasten von IPF-Patienten in vitro zu untersuchen. Sowohl die 30h-Inkubation von IPF-Fibroblasten mit LBH589 (85 nmol) als auch die mit Pirfenidon (3,0 mM) führten zu einer signifikanten Reduktion von EZM-produzierenden Genen auf mRNA- und Protein-Ebene. Ebenso konnte durch beide Behandlungen eine Supprimierung des Krebs-assoziierten Gens BIRC5 (Survivin) erreicht werden. Erwartungsgemäß wurde eine starke Acetylierung des Chromatins in LBH589- im Vergleich zu Pirfenidon- und Vehikel-behandelten Fibroblasten beobachtet, die aber auch in Pirfenidon-behandelten Zellen im Vergleich zu Vehikel signifikant erhöht war. Während LBH589 zu einer signifikanten Verminderung der Proliferation in IPF-Fibroblasten führte, die mit einer beträchtlichen Runterregulation von Proliferations-Markern (p-Histon H3, Cyclin D1) verbunden war, zeigten Pirfenidon-behandelte Zellen entgegen publizierter Studien überhaupt keine Beeinträchtigung in der Proliferation bzw. in der Expression dieser Marker. Zudem führte LBH589, nicht aber Pirfenidon, zur Induktion von ER Stress und Apoptose-Genen in IPF-Fibroblasten. Wir schlussfolgern, dass Pirfenidon neben anderer Wirkungsmechanismen auch über schwache epigenetische Veränderungen am Chromatin profibrotische Genexpression bei IPF-Patienten vermindern kann. Und wir glauben, dass pan-HDAC-Inhibitoren wie LBH589 als potentielle Wirkstoffe gegen Fibroblasten-Proliferation und Myofibroblasten-Differenzierung als alternative Therapieoption bei der IPF in Betracht gezogen werden können.

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-1β (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.

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.

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.