Novel roles of cardiac-derived erythropoietin in cardiac development and function.

The role of erythropoietin (EPO) has extended beyond hematopoiesis to include cytoprotection, inotropy, and neurogenesis. Extra-renal EPO has been reported for multiple tissue/cell types, but the physiological relevance remains unknown. Although the EPO receptor is expressed by multiple cardiac cell types and human recombinant EPO increases contractility and confers cytoprotection against injury, whether the heart produces physiologically meaningful amounts of EPO in vivo is unclear. We show a distinct circadian rhythm of cardiac EPO mRNA expression in adult mice and increased mRNA expression during embryogenesis, suggesting physiological relevance to cardiac EPO production throughout life. We then generated constitutive, cardiomyocyte-specific EPO knockout mice driven by the Mlc2v promoter (EPOfl/fl:Mlc2v-cre+/-; EPOΔ/Δ-CM). During cardiogenesis, cardiac EPO mRNA expression and cellular proliferation were reduced in EPOΔ/Δ-CM hearts. However, in adult EPOΔ/Δ- CM mice, total heart weight was preserved through increased cardiomyocyte cross-sectional area, indicating the reduced cellular proliferation was compensated for by cellular hypertrophy. Echocardiography revealed no changes in cardiac dimensions, with modest reductions in ejection fraction, stroke volume, and tachycardia, whereas invasive hemodynamics showed increased cardiac contractility and lusitropy. Paradoxically, EPO mRNA expression in the heart was elevated in adult EPOΔ/Δ-CM, along with increased serum EPO protein content and hematocrit. Using RNA fluorescent in situ hybridization, we found that Epo RNA colocalized with endothelial cells in the hearts of adult EPOΔ/Δ-CM mice, identifying the endothelial cells as a cell responsible for the EPO hyper-expression. Collectively, these data identify the first physiological roles for cardiomyocyte-derived EPO. We have established cardiac EPO mRNA expression is a complex interplay of multiple cell types, where loss of embryonic cardiomyocyte EPO production results in hyper-expression from other cells within the adult heart.

Myeloid-specific deletion of activating transcription factor 6 alpha increases CD11b+ macrophage subpopulations and aggravates lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung disease of unknown etiology. The accumulation of macrophages is associated with disease pathogenesis. The unfolded protein response (UPR) has been linked to macrophage activation in pulmonary fibrosis. To date, the impact of activating transcription factor 6 alpha (ATF6α), one of the UPR mediators, on the composition and function of pulmonary macrophage subpopulations during lung injury and fibrogenesis is not fully understood. We began by examining the expression of Atf6α in IPF patients' lung single-cell RNA sequencing dataset, archived surgical lung specimens, and CD14+ circulating monocytes. To assess the impact of ATF6α on pulmonary macrophage composition and pro-fibrotic function during tissue remodeling, we conducted an in vivo myeloid-specific deletion of Atf6α. Flow cytometric assessments of pulmonary macrophages were carried out in C57BL/6 and myeloid specific ATF6α-deficient mice in the context of bleomycin-induced lung injury. Our results demonstrated that Atf6α mRNA was expressed in pro-fibrotic macrophages found in the lung of a patient with IPF and in CD14+ circulating monocytes obtained from blood of a patient with IPF. After bleomycin administration, the myeloid-specific deletion of Atf6α altered the pulmonary macrophage composition, expanding CD11b+ subpopulations with dual polarized CD38+ CD206+ expressing macrophages. Compositional changes were associated with an aggravation of fibrogenesis including increased myofibroblast and collagen deposition. A further mechanistic ex vivo investigation revealed that ATF6α was required for CHOP induction and the death of bone marrow-derived macrophages. Overall, our findings suggest a detrimental role for the ATF6α-deficient CD11b+ macrophages which had altered function during lung injury and fibrosis.

Intrinsic BMP inhibitor Gremlin regulates alveolar epithelial type II cell proliferation and differentiation.

Type 1 alveolar epithelial cells (AT1s) and type 2 alveolar epithelial cells (AT2s) regulate the structural integrity and function of alveoli. AT1s mediate gas exchange, whereas AT2s serve multiple functions, including surfactant secretion and alveolar repair through proliferation and differentiation into AT1s as progenitors. However, mechanisms regulating AT2 proliferation and differentiation remain unclear. Here we demonstrate that Gremlin, an intrinsic inhibitor of bone morphogenetic protein (BMP), induces AT2 proliferation and differentiation. Transient overexpression of Gremlin in rat lungs by adenovirus vector delivery suppressed BMP signaling, induced proliferation of AT2s and the production of Bmp2, which in turn led to the recovery of BMP signaling and induced AT2 differentiation into AT1s. Bleomycin-induced lung injury upregulated Gremlin and showed a similar time course of biomarker expression comparable to the adenovirus model. TGF-ß and IL-1ß induced Gremlin expression in fibroblasts. Taken together, our findings implicate that Gremlin expression during lung injury leads to precisely timed inhibition of BMP signaling and activates AT2s, leading to alveolar repair.

Vascular-Parenchymal Cross-Talk Promotes Lung Fibrosis through BMPR2 Signaling.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by progressive lung scarring. IPF-related pulmonary vascular remodeling and pulmonary hypertension (PH) result in a particularly poor prognosis. Objectives: To study the pathogenesis of vascular remodeling in fibrotic lungs and its contribution to progression of fibrosis. Methods: We used an experimental model of lung fibrosis associated with PH by transient overexpression of active TGF-ß1 (transforming growth factor-ß1). Samples from patients with fibrotic lung diseases were analyzed in depth using immunostaining, gene expression, and gene mutations. Measurements and Main Results: We found a reduction in endothelial cells (ECs) and activation of vascular smooth muscle cells (VSMCs) in fibrotic lungs. Coculturing fibroblasts with VSMCs or ECs from fibrotic lungs induced fibrotic phenotypes in fibroblasts. IPF fibroblasts induced EC death and activation of VSMCs in coculture systems. Decreased concentrations of BMPR2 (bone morphogenic protein receptor 2) and its signaling were observed in ECs and VSMCs from fibrotic lungs in both rats and humans. On fibroblasts treated with media from VSMCs, BMPR2 suppression in VSMCs led to fibrogenic effects. Tacrolimus activated BMPR2 signaling and attenuated fibrosis and PH in rodent lungs. Whole-exome sequencing revealed rare mutations in PH-related genes, including BMPR2, in patients with IPF undergoing transplantation. A unique missense BMPR2 mutation (p.Q721R) was discovered to have dysfunctional effects on BMPR2 signaling. Conclusions: Endothelial dysfunction and vascular remodeling in PH secondary to pulmonary fibrosis enhance fibrogenesis through impaired BMPR2 signaling. Tacrolimus may have value as a treatment of advanced IPF and concomitant PH. Genetic abnormalities may determine the development of PH in advanced IPF.

Circulating CD200 is increased in the secretory phase of women with endometriosis as is endometrial mRNA, and endometrial stromal cell CD200R1 is increased in spite of reduced mRNA.

PROBLEM: Estrogen-dependent extrauterine implantation and growth of menstrual endometrial tissue affects roughly 10% of reproductive age women and depends on suppression of local innate immune defenses to prevent ectopic tissue rejection. Immunohistochemistry has shown the immune check-point inhibitor CD200 which can suppress rejection is expressed in eutopic endometrium and in ectopic deposits. Soluble CD200 accumulated in venules draining eutopic and ectopic endometrium of endometriosis cases in the secretory phase but not proliferative phase of the menstrual cycle, and should be increased in the circulation. METHOD OF STUDY: Sera from endometriosis and non-endometriosis controls were tested by ELISA for CD200. Endometrial CD200, CD200R1 and CD200R2 mRNA in eutopic was quantified by RT-PCR and localized by in situ hybridization. CD200R1 protein was quantified by immunohistochemistry. RESULTS: Secretory phase serum CD200 was elevated in women with endometriosis compared to controls. Serum CD200 correlated with matched endometrial CD200 mRNA levels. Expression of mRNA for CD200R1 which signals immune suppression was decreased whereas mRNA for the CD200R2 activating receptor was increased. In situ staining of CD200R1 and CD200R2 mRNA showed both receptors were expressed and the fraction of CD200R that is CD200R1 was reduced in secretory and menstrual phase endometriosis endometrium consistent with the RT-PCR result. By contrast, CD200R1 protein and CD200R1 fraction of total CD200R protein were increased in endometriosis. CONCLUSIONS: Failure to suppress circulating CD200 levels in the secretory phase had an 87% specificity and 90% sensitivity for endometriosis. CD200 and increased CD200R1 expression may facilitate development of ectopic deposits by suppressing rejection mechanisms.

Monocyte and macrophage derived myofibroblasts: Is it fate? A review of the current evidence.

Since the discovery of the myofibroblast over 50 years ago, much has been learned about its role in wound healing and fibrosis. Its origin, however, remains controversial, with a number of progenitor cells being proposed. Macrophage-myofibroblast transition (MMT) is a recent term coined in 2014 that describes the mechanism through which macrophages, derived from circulating monocytes originating in the bone marrow, transformed into myofibroblasts and contributed to kidney fibrosis. Over the past years, several studies have confirmed the existence of MMT in various systems, suggesting that MMT could potentially occur in all fibrotic conditions and constitute a reasonable therapeutic target to prevent progressive fibrotic disease. In this perspective, we examined recent evidence supporting the notion of MMT in both human disease and experimental models across organ systems. Mechanistic insight from these studies and information from in vitro studies is provided. The findings substantiating plausible MMT showcased the co-expression of macrophage and myofibroblast markers, including CD68 or F4/80 (macrophage) and α-SMA (myofibroblast), in fibroblast-like cells. Furthermore, fate-mapping experiments in murine models exhibiting myeloid-derived myofibroblasts in the tissue further provide direct evidence for MMT. Additionally, we provide some evidence from single cell RNA sequencing experiments confirmed by fluorescent in situ hybridisation studies, showing monocyte/macrophage and myofibroblast markers co-expressed in lung tissue from patients with fibrotic lung disease. In conclusion, MMT is likely a significant contributor to myofibroblast formation in wound healing and fibrotic disease across organ systems. Circulating precursors including monocytes and the molecular mechanisms governing MMT could constitute valid targets and provide insight for the development of novel antifibrotic therapies; however, further understanding of these processes is warranted.

FK506-Binding Protein 13 Expression Is Upregulated in Interstitial Lung Disease and Correlated with Clinical Severity. A Potentially Protective Role.

Pulmonary fibrosis is a progressive lung disease characterized by myofibroblast accumulation and excessive extracellular matrix deposition. We sought to investigate the role of FKBP13 (13-kD FK506-binding protein), an endoplasmic reticulum-resident molecular chaperone, in various forms of pulmonary fibrosis. We first characterized the gene and protein expression of FKBP13 in lung biopsy specimens from 24 patients with idiopathic pulmonary fibrosis and 17 control subjects. FKBP13 expression was found to be elevated in the fibrotic regions of idiopathic pulmonary fibrosis lung tissues and correlated with declining forced vital capacity and dyspnea severity. FKBP13 expression was also increased in lung biopsy specimens of patients with hypersensitivity pneumonitis, rheumatoid arthritis, and sarcoidosis-associated interstitial lung disease. We next evaluated the role of this protein using FKBP13-/- mice in a bleomycin model of pulmonary fibrosis. Animals were assessed for lung function and histopathology at different stages of lung injury including the inflammatory (Day 7), fibrotic (Day 21), and resolution (Day 50) phases. FKBP13-/- mice showed increased infiltration of inflammatory cells and cytokines at Day 7, increased lung elastance and fibrosis at Day 21, and impaired resolution of fibrosis at Day 50. These changes were associated with an increased number of cells that stained positive for TUNEL and cleaved caspase 3 in the FKBP13-/- lungs, indicating a heightened cellular sensitivity to bleomycin. Our findings suggest that FKBP13 is a potential biomarker for severity of interstitial lung diseases and that it has a biologically relevant role in protecting mice against bleomycin-induced injury, inflammation, and fibrosis.

ABCF1 Regulates dsDNA-induced Immune Responses in Human Airway Epithelial Cells.

Background: The airway epithelium represents a critical component of the human lung that helps orchestrate defenses against respiratory tract viral infections, which are responsible for more than 2.5 million deaths/year globally. Innate immune activities of the airway epithelium rely on Toll-like receptors (TLRs), nucleotide binding and leucine-rich-repeat pyrin domain containing (NLRP) receptors, and cytosolic nucleic acid sensors. ATP Binding Cassette (ABC) transporters are ubiquitous across all three domains of life-Archaea, Bacteria, and Eukarya-and expressed in the human airway epithelium. ABCF1, a unique ABC family member that lacks a transmembrane domain, has been defined as a cytosolic nucleic acid sensor that regulates CXCL10, interferon-ß expression, and downstream type I interferon responses. We tested the hypothesis that ABCF1 functions as a dsDNA nucleic acid sensor in human airway epithelial cells important in regulating antiviral responses. Methods: Expression and localization experiments were performed using in situ hybridization and immunohistochemistry in human lung tissue, while confirmatory transcript and protein expression was performed in human airway epithelial cells. Functional experiments were performed with siRNA methods in a human airway epithelial cell line. Complementary transcriptomic analyses were performed to explore the contributions of ABCF1 to gene expression patterns. Results: Using archived human lung and human airway epithelial cells, we confirm expression of ABCF1 gene and protein expression in these tissue samples, with a role for mediating CXCL10 production in response to dsDNA viral mimic challenge. Although, ABCF1 knockdown was associated with an attenuation of select genes involved in the antiviral responses, Gene Ontology analyses revealed a greater interaction of ABCF1 with TLR signaling suggesting a multifactorial role for ABCF1 in innate immunity in human airway epithelial cells. Conclusion: ABCF1 is a candidate cytosolic nucleic acid sensor and modulator of TLR signaling that is expressed at gene and protein levels in human airway epithelial cells. The precise level where ABCF1 protein functions to modulate immune responses to pathogens remains to be determined but is anticipated to involve IRF-3 and CXCL10 production.

Suboptimal treatment response to anti-IL-5 monoclonal antibodies in severe eosinophilic asthmatics with airway autoimmune phenomena.

BACKGROUND: In clinical trials, the two anti-interleukin (IL)-5 monoclonal antibodies (mAbs: mepolizumab and reslizumab) approved to treat severe eosinophilic asthma reduce exacerbations by ∼50-60%. OBJECTIVE: To observe response to anti-IL-5 mAbs in a real-life clinical setting, and to evaluate predictors of suboptimal response. METHODS: In four Canadian academic centres, predefined clinical end-points in 250 carefully characterised moderate-to-severe asthmatic patients were collected prospectively to assess response to the two anti-IL-5 mAbs. Suboptimal response was determined based on failure to reduce maintenance corticosteroid (MCS) or asthma symptoms scores (Asthma Control Questionnaire (ACQ)) or exacerbations, in addition to persistence of sputum/blood eosinophils. Worsening in suboptimal responders was assessed based on reduced lung function by 25% or increase in MCS/ACQ. A representative subset of 39 patients was evaluated for inflammatory mediators, autoantibodies and complement activation in sputum (by ELISA) and for immune-complex deposition by immunostaining formalin-fixed paraffin-embedded sputum plugs. RESULTS: Suboptimal responses were observed in 42.8% (107 out of 250) patients treated with either mepolizumab or reslizumab. Daily prednisone requirement, sinus disease and late-onset asthma diagnoses were the strongest predictors of suboptimal response. Asthma worsened in 13.6% (34 out of 250) of these patients. The majority (79%) of them were prednisone-dependent. Presence of sputum anti-eosinophil peroxidase immunoglobulin (Ig)G was a predictor of suboptimal response to an anti-IL-5 mAb. An increase in sputum C3c (marker of complement activation) and deposition of C1q-bound/IL-5-bound IgG were observed in the sputa of those patients who worsened on therapy, suggesting an underlying autoimmune-mediated pathology. CONCLUSION: A significant number of patients who meet currently approved indications for anti-IL5 mAbs show suboptimal response to them in real-life clinical practice, particularly if they are on high doses of prednisone. Monitoring blood eosinophil count is not helpful to identify these patients. The concern of worsening of symptoms associated with immune-complex mediated complement activation in a small proportion of these patients highlights the relevance of recognising airway autoimmune phenomena and this requires further evaluation.

Protein Misfolding and Endoplasmic Reticulum Stress in Chronic Lung Disease: Will Cell-Specific Targeting Be the Key to the Cure?

Chronic lung disease accounts for a significant global burden with respect to death, disability, and health-care costs. Due to the heterogeneous nature and limited treatment options for these diseases, it is imperative that the cellular and molecular mechanisms underlying the disease pathophysiology are further understood. The lung is a complex organ with a diverse cell population, and each cell type will likely have different roles in disease initiation, progression, and resolution. The effectiveness of a given therapeutic agent may depend on the net effect on each of these cell types. Over the past decade, it has been established that endoplasmic reticulum stress and the unfolded protein response are involved in the development of several chronic lung diseases. These conserved cellular pathways are important for maintaining cellular proteostasis, but their aberrant activation can result in pathology. This review discusses the current understanding of endoplasmic reticulum stress and the unfolded protein response at the cellular level in the development and progression of various chronic lung diseases. We highlight the need for increased understanding of the specific cellular contributions of unfolded protein response activation to these pathologies and suggest that the development of cell-specific targeted therapies is likely required to further decrease disease progression and to promote resolution of chronic lung disease.

IL-6 mediates ER expansion during hyperpolarization of alternatively activated macrophages.

Although recent evidence has shown that IL-6 is involved in enhanced alternative activation of macrophages toward a profibrotic phenotype, the mechanisms leading to their increased secretory capacity are not fully understood. Here, we investigated the effect of IL-6 on endoplasmic reticulum (ER) expansion and alternative activation of macrophages in vitro. An essential mediator in this ER expansion process is the IRE1 pathway, which possesses a kinase and endoribonuclease domain to cleave XBP1 into a spliced bioactive molecule. To investigate the IRE1-XBP1 expansion pathway, IL-4/IL-13 and IL-4/IL-13/IL-6-mediated alternative programming of murine bone marrow-derived and human THP1 macrophages were assessed by arginase activity in cell lysates, CD206 and arginase-1 expression by flow cytometry, and secreted CCL18 by ELISA, respectively. Ultrastructural intracellular morphology and ER biogenesis were examined by transmission electron microscopy and immunofluorescence. Transcription profiling of 128 genes were assessed by NanoString and Pharmacological inhibition of the IRE1-XBP1 arm was achieved using STF-083010 and was verified by RT-PCR. The addition of IL-6 to the conventional alternative programming cocktail IL-4/IL-13 resulted in increased ER and mitochondrial expansion, profibrotic profiles and unfolded protein response-mediated induction of molecular chaperones. IRE1-XBP1 inhibition substantially reduced the IL-6-mediated hyperpolarization and normalized the above effects. In conclusion, the addition of IL-6 enhances ER expansion and the profibrotic capacity of IL-4/IL-13-mediated activation of macrophages. Therapeutic strategies targeting IL-6 or the IRE1-XBP1 axis may be beneficial to prevent the profibrotic capacity of macrophages.