Potential Mechanisms Underlying TGF-β-mediated Complement Activation in Lung Fibrosis

While our previous studies suggest that limiting bleomycin-induced complement activation suppresses TGF-β signaling, the specific hierarchical interactions between TGF-β and complement in lung fibrosis are unclear. Herein, we investigated the mechanisms underlying TGF-β-induced complement activation in the pathogenesis of lung fibrosis. C57-BL6 mice were given intratracheal instillations of adenoviral vectors overexpressing TGF-β (Ad-TGFβ) or the firefly gene-luciferase (Ad-Luc; control). Two weeks later, mice with fibrotic lungs were instilled RNAi specific to receptors for C3a or C5a-C3ar or C5ar, and sacrificed at day 28. Histopathological analyses revealed that genetic silencing of C3ar or C5ar arrested the progression of TGF-β-induced lung fibrosis, collagen deposition and content (hydroxyproline, col1a1/2); and significantly suppressed local complement activation. With genetic silencing of either C3ar or C5ar, in Ad-TGFβ-injured lungs: we detected the recovery of Smad7 (TGF-β inhibitor) and diminished local release of DAF (membrane-bound complement inhibitor); in vitro: TGF-β-mediated loss of DAF was prevented. Conversely, blockade of the TGF-β receptor prevented C3a-mediated loss of DAF in both normal primary human alveolar and small airway epithelial cells. Of the 52 miRNAs analyzed as part of the Affymetrix array, normal primary human SAECs exposed to C3a, C5a or TGF-β caused discrete and overlapping miRNA regulation related to epithelial proliferation or apoptosis (miR-891A, miR-4442, miR-548, miR-4633), cellular contractility (miR-1197) and lung fibrosis (miR-21, miR-200C, miR-31HG, miR-503). Our studies present potential mechanisms by which TGF-β activates complement and promotes lung fibrosis

Crosstalk between TGF-β1 and complement activation augments epithelial injury in pulmonary fibrosis

The epithelial complement inhibitory proteins (CIPs) cluster of differentiation 46 and 55 (CD46 and CD55) regulate circulating immune complex-mediated complement activation in idiopathic pulmonary fibrosis (IPF). Our previous studies demonstrated that IL-17A mediates epithelial injury via transforming growth factor β1 (TGF-β1) and down-regulates CIPs. In the current study, we examined the mechanistic role of TGF-β1 in complement activation-mediated airway epithelial injury in IPF pathogenesis. We observed lower epithelial CIP expression in IPF lungs compared to normal lungs, associated with elevated levels of complement component 3a and 5a (C3a and C5a), locally and systemically. In normal primary human small airway epithelial cells (SAECs) treated with TGF-β1 (10 ng/ml), C3a, or C5a (100 nM), we observed loss of CIPs and increased poly(ADP-ribose) polymerase (PARP) activation [also observed with RNA interference (RNAi) of CD46/CD55]. TGF-β1-mediated loss of CIPs and Snail induction [SNAI1; a transcriptional repressor of E-cadherin (E-CAD)] was blocked by inhibiting mitogen-activated protein kinase (p38MAPK; SB203580) and RNAi silencing of SNAI1. C3a- and C5a-mediated loss of CIPs was also blocked by p38MAPK inhibition. While C3a upregulated TGFb transcripts, both C3a and C5a down-regulated SMAD7 (negative regulator of TGF-β), and whereas TGF-β1 induced C3a/C5a receptor (C3aR/C5aR) expression, pharmacologic C3aR/C5aR inhibition protected against C3a-/C5a-mediated loss of CIPs. Taken together, our results suggest that epithelial injury in IPF can be collectively amplified as a result of TGF-β1-induced loss of CIPs leading to complement activation that down-regulates CIPs and induces TGF-β1 expressio

IL‐17A deficiency mitigates bleomycin‐induced complement activation during lung fibrosis

Interleukin 17A (IL‐17A) and complement (C′) activation have each been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). We have reported that IL‐17A induces epithelial injury via TGF‐β in murine bronchiolitis obliterans; that TGF‐β and the C′ cascade present signaling interactions in mediating epithelial injury; and that the blockade of C′ receptors mitigates lung fibrosis. In the present study, we investigated the role of IL‐17A in regulating C′ in lung fibrosis. Microarray analyses of mRNA isolated from primary normal human small airway epithelial cells indicated that IL‐17A (100 ng/ml; 24 h; n = 5 donor lungs) induces C′ components (C′ factor B, C3, and GPCR kinase isoform 5), cytokines (IL8, ‐6, and ‐1B), and cytokine ligands (CXCL1, ‐2, ‐3, ‐5, ‐6, and ‐16). IL‐17A induces protein and mRNA regulation of C′ components and the synthesis of active C′ 3a (C3a) in normal primary human alveolar type II epithelial cells (AECs). Wild‐type mice subjected to IL‐17A neutralization and IL‐17A knockout (i717a−/−) mice were protected against bleomycin (BLEO)‐induced fibrosis and collagen deposition. Further, BLEO‐injured i17a−/− mice had diminished levels of circulating Krebs Von Den Lungen 6 (alveolar epithelial injury marker), local caspase‐3/7, and local endoplasmic reticular stress‐related genes. BLEO‐induced local C′ activation [C3a, C5a, and terminal C′ complex (C5b‐9)] was attenuated in il17a−/− mice, and IL‐17A neutralization prevented the loss of epithelial C′ inhibitors (C′ receptor‐1 related isoform Y and decay accelerating factor), and an increase in local TUNEL levels. RNAi‐mediated gene silencing of il17a in fibrotic mice arrested the progression of lung fibrosis, attenuated cellular apoptosis (caspase‐3/7) and lung deposition of collagen and C′ (C5b‐9). Compared to normals, plasma from IPF patients showed significantly higher hemolytic activity. Our findings demonstrate that limiting complement activation by neutralizing IL‐17A is a potential mechanism in ameliorating lung fibrosis.—Cipolla, E., Fisher, A. J., Gu, H., Mickler, E. A., Agarwal, M., Wilke, C. A., Kim, K. K., Moore, B. B., Vittal, R. IL‐17A deficiency mitigates bleomycin‐induced complement activation during lung fibrosis. FASEB J. 31, 5543–5556 (2017). www.fasebj.orgPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154482/1/fsb2fj201700289r-sup-0001.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154482/2/fsb2fj201700289r.pd

Role of Complement Activation in Obliterative Bronchiolitis Post Lung Transplantation

Obliterative bronchiolitis (OB) post lung transplantation involves IL-17 regulated autoimmunity to type V collagen and alloimmunity, which could be enhanced by complement activation. However, the specific role of complement activation in lung allograft pathology, IL-17 production, and OB are unknown. The current study examines the role of complement activation in OB. Complement regulatory protein (CRP) (CD55, CD46, Crry/CD46) expression was down regulated in human and murine OB; and C3a, a marker of complement activation, was up regulated locally. IL-17 differentially suppressed Crry expression in airway epithelial cells in vitro. Neutralizing IL-17 recovered CRP expression in murine lung allografts and decreased local C3a production. Exogenous C3a enhanced IL-17 production from alloantigen or autoantigen (type V collagen) reactive lymphocytes. Systemically neutralizing C5 abrogated the development of OB, reduced acute rejection severity, lowered systemic and local levels of C3a and C5a, recovered CRP expression, and diminished systemic IL-17 and IL-6 levels. These data indicated that OB induction is in part complement dependent due to IL-17 mediated down regulation of CRPs on airway epithelium. C3a and IL-17 are part of a feed forward loop that may enhance CRP down regulation, suggesting that complement blockade could be a therapeutic strategy for OB

The HMGB1-RAGE axis mediates traumatic brain injury-induced pulmonary dysfunction in lung transplantation

Traumatic brain injury (TBI) results in systemic inflammatory responses that affect the lung. This is especially critical in the setting of lung transplantation, where more than half of donor allografts are obtained postmortem from individuals with TBI. The mechanism by which TBI causes pulmonary dysfunction remains unclear but may involve the interaction of high-mobility group box-1 (HMGB1) protein with the receptor for advanced glycation end products (RAGE). To investigate the role of HMGB1 and RAGE in TBI-induced lung dysfunction, RAGE-sufficient (wild-type) or RAGE-deficient (RAGE(-/-)) C57BL/6 mice were subjected to TBI through controlled cortical impact and studied for cardiopulmonary injury. Compared to control animals, TBI induced systemic hypoxia, acute lung injury, pulmonary neutrophilia, and decreased compliance (a measure of the lungs' ability to expand), all of which were attenuated in RAGE(-/-) mice. Neutralizing systemic HMGB1 induced by TBI reversed hypoxia and improved lung compliance. Compared to wild-type donors, lungs from RAGE(-/-) TBI donors did not develop acute lung injury after transplantation. In a study of clinical transplantation, elevated systemic HMGB1 in donors correlated with impaired systemic oxygenation of the donor lung before transplantation and predicted impaired oxygenation after transplantation. These data suggest that the HMGB1-RAGE axis plays a role in the mechanism by which TBI induces lung dysfunction and that targeting this pathway before transplant may improve recipient outcomes after lung transplantation

Effects of the protein kinase inhibitor, imatinib mesylate, on epithelial/mesenchymal phenotypes: implications for treatment of fibrotic diseases

Tissue injury in mammals triggers both inflammatory and repair responses that, in some contexts, results in fibrosis. Fibrosis is characterized by the persistence of activated myofibroblasts, ineffective re-epithelialization, and variable degrees of inflam-mation within injured tissues. The protein kinase inhibitor (PKI), imatinib mesylate, has been proposed as a potential antifibrotic therapeutic agent. In this study, the efficacy of imatinib mesy-late to modulate fibrogenic responses, both in vitro and in vivo, was examined. In an in vitro fibroblast culture model, imatinib inhibits platelet-derived growth factor receptor activation and fibroblast proliferation but not the stably differentiated myofi-broblast phenotype. Furthermore, imatinib inhibits lung epithe-lial cell proliferation and survival but not the induction of epithelial-mesenchymal transition. Imatinib does not alte

Ossabaw Pig Demonstrates Detrusor Fibrosis and Detrusor Underactivity Associated with Oxidative Stress in Metabolic Syndrome

Metabolic Syndrome (MetS) has detrimental effects on the bladder, including detrusor underactivity. The progression and mechanism of disease are poorly understood. A swine model for diabetic bladder dysfunction (DBD) was established because of the pig's human-sized bladder and its ability to develop MetS by dietary modification alone. The hypothesis of this study is that this swine model will demonstrate oxidative stress associated with MetS, which contributes to both bladder fibrosis and detrusor underactivity (DU). Ossabaw pigs underwent dietary modification consisting of a hypercaloric, atherogenic diet for 10 mo to induce MetS, and were compared with a group of control (lean) pigs. Urodynamic studies were performed in both groups to confirm DU. Thiobarbituric acid reactive substances (TBARS) detected in the urine were used to measure oxidative stress activity in the urinary tract, and urinary IL17a was used to detect profibrotic activity. MetS was confirmed by assessing body weight, blood pressure, glucose tolerance, total cholesterol, and triglycerides. The MetS group exhibited an increase in the relative levels of urinary TBARS and IL17a. Bladder pressures at capacity were lower in the MetS group, suggesting DU. Histologic analysis of a cohort of control (lean) and MetS pigs revealed that as compared with the control pigs, the MetS pigs had significantly more collagen in the muscularis layer, but not in the submucosa or mucosa layer. In conclusion, the Ossabaw pig model for diet-induced MetS is associated with oxidative stress and profibrotic activity in the bladder, which results in DU. This has previously been shown in mice and rats, but never in pigs. This novel model will better represent human MetS and DBD because the mechanism and size of the pig bladder more closely resemble that of a human, resulting in a more valid model and facilitating further study into the signaling mechanisms responsible for this impairment

Interleukin 6 trans‐signaling is a critical driver of lung allograft fibrosis

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/168372/1/ajt16417.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/168372/2/ajt16417_am.pd

Humoral immune responses mediate the development of a restrictive phenotype of chronic lung allograft dysfunction

Understanding the distinct pathogenic mechanisms that culminate in allograft fibrosis and chronic graft failure is key in improving outcomes after solid organ transplantation. Here, we describe an F1 → parent orthotopic lung transplant model of restrictive allograft syndrome (RAS), a particularly fulminant form of chronic lung allograft dysfunction (CLAD), and identify a requisite pathogenic role for humoral immune responses in development of RAS. B6D2F1/J (H2-b/d) donor lungs transplanted into the parent C57BL/6J (H2-b) recipients demonstrated a spectrum of histopathologic changes, ranging from lymphocytic infiltration, fibrinous exudates, and endothelialitis to peribronchial and pleuroparenchymal fibrosis, similar to those noted in the human RAS lungs. Gene expression profiling revealed differential humoral immune cell activation as a key feature of the RAS murine model, with significant B cell and plasma cell infiltration noted in the RAS lung allografts. B6D2F1/J lung allografts transplanted into μMt-/- (mature B cell deficient) or activation-induced cytidine deaminase (AID)/secretory μ-chain (μs) double-KO (AID-/-μs-/-) C57BL/6J mice demonstrated significantly decreased allograft fibrosis, indicating a key role for antibody secretion by B cells in mediating RAS pathology. Our study suggests that skewing of immune responses determines the diverse allograft remodeling patterns and highlights the need to develop targeted therapies for specific CLAD phenotypes