Reduced Bik expression drives low-grade airway inflammation and increased risk for COPD in females.

Chronic low-grade inflammation is increasingly recognized as a subtle yet potent risk factor for a multitude of age-related disorders, including respiratory diseases, cardiovascular conditions, metabolic syndromes, autoimmunity, and cancer. In this issue of the JCI, Mebratu, Jones, and colleagues shed new light on the mechanisms that promote low-grade airway inflammation and how this contributes to the development of chronic obstructive pulmonary disease (COPD). Their finding that Bik deficiency leads to spontaneous emphysema in female mice, but not in males, marks a notable advancement in our understanding of how inflammatory processes can diverge based on biological sex. This finding is of clinical relevance, given the vulnerability of women to developing COPD.

Regulation of epithelial transitional states in murine and human pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease arising from impaired regeneration of the alveolar epithelium after injury. During regeneration, type 2 alveolar epithelial cells (AEC2s) assume a transitional state that upregulates multiple keratins and ultimately differentiate into AEC1s. In IPF, transitional AECs accumulate with ineffectual AEC1 differentiation. However, whether and how transitional cells cause fibrosis, whether keratins regulate transitional cell accumulation and fibrosis, and why transitional AECs and fibrosis resolve in mouse models but accumulate in IPF are unclear. Here, we show that human keratin 8 (KRT8) genetic variants were associated with IPF. Krt8-/- mice were protected from fibrosis and accumulation of the transitional state. Keratin 8 (K8) regulated the expression of macrophage chemokines and macrophage recruitment. Profibrotic macrophages and myofibroblasts promoted the accumulation of transitional AECs, establishing a K8-dependent positive feedback loop driving fibrogenesis. Finally, rare murine transitional AECs were highly senescent and basaloid and may not differentiate into AEC1s, recapitulating the aberrant basaloid state in human IPF. We conclude that transitional AECs induced and were maintained by fibrosis in a K8-dependent manner; in mice, most transitional cells and fibrosis resolved, whereas in human IPF, transitional AECs evolved into an aberrant basaloid state that persisted with progressive fibrosis.

Inhibition of antiapoptotic BCL-2 proteins with ABT-263 induces fibroblast apoptosis, reversing persistent pulmonary fibrosis.

Patients with progressive fibrosing interstitial lung diseases (PF-ILDs) carry a poor prognosis and have limited therapeutic options. A hallmark feature is fibroblast resistance to apoptosis, leading to their persistence, accumulation, and excessive deposition of extracellular matrix. A complex balance of the B cell lymphoma 2 (BCL-2) protein family controlling the intrinsic pathway of apoptosis and fibroblast reliance on antiapoptotic proteins has been hypothesized to contribute to this resistant phenotype. Examination of lung tissue from patients with PF-ILD (idiopathic pulmonary fibrosis and silicosis) and mice with PF-ILD (repetitive bleomycin and silicosis) showed increased expression of antiapoptotic BCL-2 family members in α-smooth muscle actin-positive fibroblasts, suggesting that fibroblasts from fibrotic lungs may exhibit increased susceptibility to inhibition of antiapoptotic BCL-2 family members BCL-2, BCL-XL, and BCL-W with the BH3 mimetic ABT-263. We used 2 murine models of PF-ILD to test the efficacy of ABT-263 in reversing established persistent pulmonary fibrosis. Treatment with ABT-263 induced fibroblast apoptosis, decreased fibroblast numbers, and reduced lung collagen levels, radiographic disease, and histologically evident fibrosis. Our studies provide insight into how fibroblasts gain resistance to apoptosis and become sensitive to the therapeutic inhibition of antiapoptotic proteins. By targeting profibrotic fibroblasts, ABT-263 offers a promising therapeutic option for PF-ILDs.

Alveolar epithelial cells and microenvironmental stiffness synergistically drive fibroblast activation in three-dimensional hydrogel lung models.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease that progressively and irreversibly alters the lung parenchyma, eventually leading to respiratory failure. The study of this disease has been historically challenging due to the myriad of complex processes that contribute to fibrogenesis and the inherent difficulty in accurately recreating the human pulmonary environment in vitro. Here, we describe a poly(ethylene glycol) PEG hydrogel-based three-dimensional model for the co-culture of primary murine pulmonary fibroblasts and alveolar epithelial cells that reproduces the micro-architecture, cell placement, and mechanical properties of healthy and fibrotic lung tissue. Co-cultured cells retained normal levels of viability up to at least three weeks and displayed differentiation patterns observed in vivo during IPF progression. Interrogation of protein and gene expression within this model showed that myofibroblast activation required both extracellular mechanical cues and the presence of alveolar epithelial cells. Differences in gene expression indicated that cellular co-culture induced TGF-ß signaling and proliferative gene expression, while microenvironmental stiffness upregulated the expression of genes related to cell-ECM interactions. This biomaterial-based cell culture system serves as a significant step forward in the accurate recapitulation of human lung tissue in vitro and highlights the need to incorporate multiple factors that work together synergistically in vivo into models of lung biology of health and disease.

Engineering Hybrid-Hydrogels Comprised of Healthy or Diseased Decellularized Extracellular Matrix to Study Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis is a chronic disease characterized by progressive lung scarring that inhibits gas exchange. Evidence suggests fibroblast-matrix interactions are a prominent driver of disease. However, available preclinical models limit our ability to study these interactions. We present a technique for synthesizing phototunable poly(ethylene glycol) (PEG)-based hybrid-hydrogels comprising healthy or fibrotic decellularized extracellular matrix (dECM) to decouple mechanical properties from composition and elucidate their roles in fibroblast activation. Here, we engineered and characterized phototunable hybrid-hydrogels using molecular techniques such as ninhydrin and Ellman's assays to assess dECM functionalization, and parallel-plate rheology to measure hydrogel mechanical properties. These biomaterials were employed to investigate the activation of fibroblasts from dual-transgenic Col1a1-GFP and αSMA-RFP reporter mice in response to changes in composition and mechanical properties. We show that reacting functionalized dECM from healthy or bleomycin-injured mouse lungs with PEG alpha-methacrylate (αMA) in an off-stoichiometry Michael-addition reaction created soft hydrogels mimicking a healthy lung elastic modulus (4.99 ± 0.98 kPa). Photoinitiated stiffening increased the material modulus to fibrotic values (11.48 ± 1.80 kPa). Percent activation of primary murine fibroblasts expressing Col1a1 and αSMA increased by approximately 40% following dynamic stiffening of both healthy and bleomycin hybrid-hydrogels. There were no significant differences between fibroblast activation on stiffened healthy versus stiffened bleomycin-injured hybrid-hydrogels. Phototunable hybrid-hydrogels provide an important platform for probing cell-matrix interactions and developing a deeper understanding of fibrotic activation in pulmonary fibrosis. Our results suggest that mechanical properties are a more significant contributor to fibroblast activation than biochemical composition within the scope of the hybrid-hydrogel platform evaluated in this study. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00726-y.

New Insights into Clinical and Mechanistic Heterogeneity of the Acute Respiratory Distress Syndrome: Summary of the Aspen Lung Conference 2021.

Clinical and molecular heterogeneity are common features of human disease. Understanding the basis for heterogeneity has led to major advances in therapy for many cancers and pulmonary diseases such as cystic fibrosis and asthma. Although heterogeneity of risk factors, disease severity, and outcomes in survivors are common features of the acute respiratory distress syndrome (ARDS), many challenges exist in understanding the clinical and molecular basis for disease heterogeneity and using heterogeneity to tailor therapy for individual patients. This report summarizes the proceedings of the 2021 Aspen Lung Conference, which was organized to review key issues related to understanding clinical and molecular heterogeneity in ARDS. The goals were to review new information about ARDS phenotypes, to explore multicellular and multisystem mechanisms responsible for heterogeneity, and to review how best to account for clinical and molecular heterogeneity in clinical trial design and assessment of outcomes. The report concludes with recommendations for future research to understand the clinical and basic mechanisms underlying heterogeneity in ARDS to advance the development of new treatments for this life-threatening critical illness.

Opportunities to improve nitrogen use efficiency in an intensive vegetable system without compromising yield.

Intensive vegetable cropping systems rely heavily on nitrogen (N) inputs from multiple synthetic and organic fertilizer applications. The majority of applied N is lost to the environment through numerous pathways, including as nitrous oxide (N2 O). A field trial was conducted to examine the opportunities to reduce N input in an intensive vegetable system without compromising yield. Treatments applied were control (no N), manure (M, 408 kg N ha-1 from chicken manure), grower practice (GP, 408 kg N ha-1 from chicken manure + 195 kg N ha-1 from fertilizer), and 2/3 GP (two-thirds of the total N input in GP), all with and without 3,4-dimethylpyrazole phosphate (DMPP). Nitrogen recovery in the GP treatment was determined using 15 N-labeled fertilizer. Using only manure significantly lowered celery (Apium graveolens L.) yield and apparent N use efficiency (ANUE) compared with GP. Reducing N input by one-third did not affect yield or ANUE. Use of DMPP increased ANUE despite no yield improvement. More than 50% of the applied N in the GP treatment was lost to the environment, with almost 10 kg N ha-1 emitted as N2 O over the season, which was 67 times more than from the control. Reducing the N input by one-third or using manure only reduced N2 O emissions by more than 70% relative to GP. This study shows that there is a clear opportunity to reduce N input and N2 O emissions in high-fertilizer-input vegetable systems without compromising vegetable yield.

Persistent, Progressive Pulmonary Fibrosis and Epithelial Remodeling in Mice.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic interstitial lung disease with underlying mechanisms that have been primarily investigated in mice after intratracheal instillation of a single dose of bleomycin. However, the model has significant limitations, including transient fibrosis that spontaneously resolves and its failure to fully recapitulate the epithelial remodeling in the lungs of patients with IPF. Thus, there remains an unmet need for a preclinical model with features that more closely resemble the human disease. Repetitive intratracheal instillation of bleomycin has previously been shown to recapitulate some of these features, but the instillation procedure is complex, and the long-term consequences on epithelial remodeling and fibrosis persistence and progression remain poorly understood. Here, we developed a simplified repetitive bleomycin instillation strategy consisting of three bi-weekly instillations that leads to persistent and progressive pulmonary fibrosis. Lung histology demonstrates increased collagen deposition, fibroblast accumulation, loss of type I and type II alveolar epithelial cells within fibrotic areas, bronchiolization of the lung parenchyma with CCSP+ cells, remodeling of the distal lung into cysts reminiscent of simple honeycombing, and accumulation of hyperplastic transitional KRT8+ epithelial cells. Micro-computed tomographic imaging demonstrated significant traction bronchiectasis and subpleural fibrosis. Thus, the simplified repetitive bleomycin instillation strategy leads to progressive fibrosis and recapitulates the histological and radiographic characteristics of IPF. Compared with the single bleomycin instillation model, we suggest that the simplified repetitive instillation model may be better suited to address mechanistic questions about IPF pathogenesis and preclinical studies of antifibrotic drug candidates.

Loss of Fas signaling in fibroblasts impairs homeostatic fibrosis resolution and promotes persistent pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible fibrotic disease of the distal lung alveoli that culminates in respiratory failure and reduced lifespan. Unlike normal lung repair in response to injury, IPF is associated with the accumulation and persistence of fibroblasts and myofibroblasts, as well as continued production of collagen and other extracellular matrix (ECM) components. Prior in vitro studies have led to the hypothesis that the development of resistance to Fas-induced apoptosis by lung fibroblasts and myofibroblasts contributes to their accumulation in the distal lung tissues of IPF patients. Here, we test this hypothesis in vivo in the resolving model of bleomycin-induced pulmonary fibrosis in mice. Using genetic loss-of-function approaches to inhibit Fas signaling in fibroblasts, potentially novel flow cytometry strategies to quantify lung fibroblast subsets, and transcriptional profiling of lung fibroblasts by bulk and single cell RNA sequencing, we show that Fas is necessary for lung fibroblast apoptosis during homeostatic resolution of bleomycin-induced pulmonary fibrosis in vivo. Furthermore, we show that loss of Fas signaling leads to the persistence and continued profibrotic functions of lung fibroblasts. Our studies provide insights into the mechanisms that contribute to fibroblast survival, persistence, and continued ECM deposition in the context of IPF and how failure to undergo Fas-induced apoptosis impairs fibrosis resolution.

Protein tyrosine phosphatase-α amplifies transforming growth factor-ß-dependent profibrotic signaling in lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a progressive, often fatal, fibrosing lung disease for which treatment remains suboptimal. Fibrogenic cytokines, including transforming growth factor-ß (TGF-ß), are central to its pathogenesis. Protein tyrosine phosphatase-α (PTPα) has emerged as a key regulator of fibrogenic signaling in fibroblasts. We have reported that mice globally deficient in PTPα (Ptpra-/-) were protected from experimental pulmonary fibrosis, in part via alterations in TGF-ß signaling. The goal of this study was to determine the lung cell types and mechanisms by which PTPα controls fibrogenic pathways and whether these pathways are relevant to human disease. Immunohistochemical analysis of lungs from patients with IPF revealed that PTPα was highly expressed by mesenchymal cells in fibroblastic foci and by airway and alveolar epithelial cells. To determine whether PTPα promotes profibrotic signaling pathways in lung fibroblasts and/or epithelial cells, we generated mice with conditional (floxed) Ptpra alleles (Ptpraf/f). These mice were crossed with Dermo1-Cre or with Sftpc-CreERT2 mice to delete Ptpra in mesenchymal cells and alveolar type II cells, respectively. Dermo1-Cre/Ptpraf/f mice were protected from bleomycin-induced pulmonary fibrosis, whereas Sftpc-CreERT2/Ptpraf/f mice developed pulmonary fibrosis equivalent to controls. Both canonical and noncanonical TGF-ß signaling and downstream TGF-ß-induced fibrogenic responses were attenuated in isolated Ptpra-/- compared with wild-type fibroblasts. Furthermore, TGF-ß-induced tyrosine phosphorylation of TGF-ß type II receptor and of PTPα were attenuated in Ptpra-/- compared with wild-type fibroblasts. The phenotype of cells genetically deficient in PTPα was recapitulated with the use of a Src inhibitor. These findings suggest that PTPα amplifies profibrotic TGF-ß-dependent pathway signaling in lung fibroblasts.

Soil greenhouse gas emissions from Australian sports fields.

Managed turf is a potential net source of greenhouse gas (GHG) emissions. While most studies to date have focused on non-sports turf, sports turf may pose an even greater risk of high GHG emissions due to the generally more intensive fertiliser, irrigation and mowing regimes. This study used manual and automated chambers to measure nitrous oxide (N2O) and methane (CH4) emissions from three sports fields and an area of non-sports turf in southern Australia. Over 213 days (autumn to late spring), the average daily N2O emission was 37.6 g N ha-1day-1 at a sports field monitored at least weekly and cumulative N2O emission was 2.5 times higher than the adjacent non-sports turf. Less frequent seasonal sampling at two other sports fields showed average N2O daily emission ranging from 26 to 90 g N ha-1 day-1. Management practices associated with periods of relatively high N2O emissions were surface renovation and herbicide application. CH4 emissions at all of the sports fields were generally negligible with the exception of brief periods when soil was waterlogged following heavy rainfall where emissions of up to 1.3 kg C ha-1 day-1 were recorded. Controlled release and nitrification inhibitor containing fertilisers didn't reduce N2O, CH4 or CO2 emissions relative to urea in a short term experiment. The N2O emissions from the sports fields, and even the lower emissions from the non-sports turf, were relatively high compared to other land uses in Australia highlighting the importance of accounting for these emissions at a national level and investigating mitigation practices.

Potential of nintedanib in treatment of progressive fibrosing interstitial lung diseases.

A proportion of patients with fibrosing interstitial lung diseases (ILDs) develop a progressive phenotype characterised by decline in lung function, worsening quality of life and early mortality. Other than idiopathic pulmonary fibrosis (IPF), there are no approved drugs for fibrosing ILDs and a poor evidence base to support current treatments. Fibrosing ILDs with a progressive phenotype show commonalities in clinical behaviour and in the pathogenic mechanisms that drive disease worsening. Nintedanib is an intracellular inhibitor of tyrosine kinases that has been approved for treatment of IPF and has recently been shown to reduce the rate of lung function decline in patients with ILD associated with systemic sclerosis (SSc-ILD). In vitro data demonstrate that nintedanib inhibits several steps in the initiation and progression of lung fibrosis, including the release of pro-inflammatory and pro-fibrotic mediators, migration and differentiation of fibrocytes and fibroblasts, and deposition of extracellular matrix. Nintedanib also inhibits the proliferation of vascular cells. Studies in animal models with features of fibrosing ILDs such as IPF, SSc-ILD, rheumatoid arthritis-ILD, hypersensitivity pneumonitis and silicosis demonstrate that nintedanib has anti-fibrotic activity irrespective of the trigger for the lung pathology. This suggests that nintedanib inhibits fundamental processes in the pathogenesis of fibrosis. A trial of nintedanib in patients with progressive fibrosing ILDs other than IPF (INBUILD) will report results in 2019.

FGF10-FGFR2B Signaling Generates Basal Cells and Drives Alveolar Epithelial Regeneration by Bronchial Epithelial Stem Cells after Lung Injury.

Idiopathic pulmonary fibrosis is a common form of interstitial lung disease resulting in alveolar remodeling and progressive loss of pulmonary function because of chronic alveolar injury and failure to regenerate the respiratory epithelium. Histologically, fibrotic lesions and honeycomb structures expressing atypical proximal airway epithelial markers replace alveolar structures, the latter normally lined by alveolar type 1 (AT1) and AT2 cells. Bronchial epithelial stem cells (BESCs) can give rise to AT2 and AT1 cells or honeycomb cysts following bleomycin-mediated lung injury. However, little is known about what controls this binary decision or whether this decision can be reversed. Here we report that inactivation of Fgfr2b in BESCs impairs their contribution to both alveolar epithelial regeneration and honeycomb cysts after bleomycin injury. By contrast overexpression of Fgf10 in BESCs enhances fibrosis resolution by favoring the more desirable outcome of alveolar epithelial regeneration over the development of pathologic honeycomb cysts.

Overview of Innate Lung Immunity and Inflammation.

The nasal passages, conducting airways and gas-exchange surfaces of the lung, are constantly exposed to substances contained in the air that we breathe. While many of these suspended substances are relatively harmless, some, for example, pathogenic microbes, noxious pollutants, and aspirated gastric contents can be harmful. The innate immune system, lungs and conducting airways have evolved specialized mechanisms to protect the respiratory system not only from these harmful inhaled substances but also from the overly exuberant innate immune activation that can arise during the host response to harmful inhaled substances. Herein, we discuss the cell types that contribute to lung innate immunity and inflammation and how their activities are coordinated to promote lung health.

Protein Tyrosine Phosphatase-N13 Promotes Myofibroblast Resistance to Apoptosis in Idiopathic Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a progressive, fibrotic interstitial lung disease characterized by (myo)fibroblast accumulation and collagen deposition. Resistance to Fas-induced apoptosis is thought to facilitate (myo)fibroblast persistence in fibrotic lung tissues by poorly understood mechanisms. OBJECTIVES: To test the hypothesis that PTPN13 (protein tyrosine phosphatase-N13) is expressed by IPF lung (myo)fibroblasts, promotes their resistance to Fas-induced apoptosis, and contributes to the development of pulmonary fibrosis. METHODS: PTPN13 was localized in lung tissues from patients with IPF and control subjects by immunohistochemical staining. Inhibition of PTPN13 function in primary IPF and normal lung (myo)fibroblasts was accomplished by: 1) downregulation with TNF-α (tumor necrosis factor-α)/IFN-γ, 2) siRNA knockdown, or 3) a cell-permeable Fas/PTPN13 interaction inhibitory peptide. The role of PTPN13 in the development of pulmonary fibrosis was assessed in mice with genetic deficiency of PTP-BL, the murine ortholog of PTPN13. MEASUREMENTS AND MAIN RESULTS: PTPN13 was constitutively expressed by (myo)fibroblasts in the fibroblastic foci of patients with IPF. Human lung (myo)fibroblasts, which are resistant to Fas-induced apoptosis, basally expressed PTPN13 in vitro. TNF-α/IFN-γ or siRNA-mediated PTPN13 downregulation and peptide-mediated inhibition of the Fas/PTPN13 interaction in human lung (myo)fibroblasts promoted Fas-induced apoptosis. Bleomycin-challenged PTP-BL-/- mice, while developing inflammatory lung injury, exhibited reduced pulmonary fibrosis compared with wild-type mice. CONCLUSIONS: These findings suggest that PTPN13 mediates the resistance of human lung (myo)fibroblasts to Fas-induced apoptosis and promotes pulmonary fibrosis in mice. Our results suggest that strategies aimed at interfering with PTPN13 expression or function may represent a novel strategy to reduce fibrosis in IPF.

Nintedanib reduces pulmonary fibrosis in a model of rheumatoid arthritis-associated interstitial lung disease.

Rheumatoid arthritis (RA)-associated interstitial lung disease (RA-ILD) develops in ~20% of patients with RA. SKG mice, which are genetically prone to development of autoimmune arthritis, develop a pulmonary interstitial pneumonia that resembles human cellular and fibrotic nonspecific interstitial pneumonia. Nintedanib, a tyrosine kinase inhibitor approved for treatment of idiopathic pulmonary fibrosis, has been shown to reduce the decline in lung function. Therefore, we investigated the effect of nintedanib on development of pulmonary fibrosis and joint disease in female SKG mice with arthritis induced by intraperitoneal injection of zymosan (5 mg). Nintedanib (60 mg·kg-1·day-1 via oral gavage) was started 5 or 10 wk after injection of zymosan. Arthritis and lung fibrosis outcome measures were assessed after 6 wk of treatment with nintedanib. A significant reduction in lung collagen levels, determined by measuring hydroxyproline levels and staining for collagen, was observed after 6 wk in nintedanib-treated mice with established arthritis and lung disease. Early intervention with nintedanib significantly reduced development of arthritis based on joint assessment and high-resolution µ-CT. This study impacts the RA and ILD fields by facilitating identification of a therapeutic treatment that may improve both diseases. As this model replicates the characteristics of RA-ILD, the results may be translatable to the human disease.

Hypoxia-Inducible Factor 1α Signaling Promotes Repair of the Alveolar Epithelium after Acute Lung Injury.

During the acute respiratory distress syndrome, epithelial cells, primarily alveolar type (AT) I cells, die and slough off, resulting in enhanced permeability. ATII cells proliferate and spread onto the denuded basement membrane to reseal the barrier. Repair of the alveolar epithelium is critical for clinical recovery; however, mechanisms underlying ATII cell proliferation and spreading are not well understood. We hypothesized that hypoxia-inducible factor (HIF)1α promotes proliferation and spreading of ATII cells during repair after lung injury. Mice were treated with lipopolysaccharide or hydrochloric acid. HIF activation in ATII cells after injury was demonstrated by increased luciferase activity in oxygen degradation domain-Luc (HIF reporter) mice and expression of the HIF1α target gene GLUT1. ATII cell proliferation during repair was attenuated in ATII cell-specific HIF1α knockout (SftpcCreERT2+/-;HIF1αf/f) mice. The HIF target vascular endothelial growth factor promoted ATII cell proliferation in vitro and after lung injury in vivo. In the scratch wound assay of cell spreading, HIF stabilization accelerated, whereas HIF1α shRNA delayed wound closure. SDF1 and its receptor, CXCR4, were found to be HIF1α-regulated genes in ATII cells and were up-regulated during lung injury. Stromal cell-derived factor 1/CXCR4 inhibition impaired cell spreading and delayed the resolution of permeability after lung injury. We conclude that HIF1α is activated in ATII cells after lung injury and promotes proliferation and spreading during repair.

Nitrification inhibitors can increase post-harvest nitrous oxide emissions in an intensive vegetable production system.

To investigate the effect of nitrification inhibitors (NIs) 3,4-dimethylpyrazole phosphate (DMPP) and 3-methylpyrazole 1,2,4-triazole (3MP + TZ), on N2O emissions and yield from a typical vegetable rotation in sub-tropical Australia we monitored soil N2O fluxes continuously over an entire year using an automated greenhouse gas measurement system. The temporal variation of N2O fluxes showed only low emissions over the vegetable cropping phases, but significantly higher emissions were observed post-harvest accounting for 50-70% of the annual emissions. NIs reduced N2O emissions by 20-60% over the vegetable cropping phases; however, this mitigation was offset by elevated N2O emissions from the NIs treatments over the post-harvest fallow period. Annual N2O emissions from the conventional fertiliser, the DMPP treatment, and the 3MP + TZ treatment were 1.3, 1.1 and 1.6 (sem = 0.2) kg-N ha-1 year-1, respectively. This study highlights that the use of NIs in vegetable systems can lead to elevated N2O emissions by storing N in the soil profile that is available to soil microbes during the decomposition of the vegetable residues. Hence the use of NIs in vegetable systems has to be treated carefully and fertiliser rates need to be adjusted to avoid an oversupply of N during the post-harvest phase.

Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs.

Mesenchymal stem cells (MSCs) and macrophages are fundamental components of the stem cell niche and function coordinately to regulate haematopoietic stem cell self-renewal and mobilization. Recent studies indicate that mitophagy and healthy mitochondrial function are critical to the survival of stem cells, but how these processes are regulated in MSCs is unknown. Here we show that MSCs manage intracellular oxidative stress by targeting depolarized mitochondria to the plasma membrane via arrestin domain-containing protein 1-mediated microvesicles. The vesicles are then engulfed and re-utilized via a process involving fusion by macrophages, resulting in enhanced bioenergetics. Furthermore, we show that MSCs simultaneously shed micro RNA-containing exosomes that inhibit macrophage activation by suppressing Toll-like receptor signalling, thereby de-sensitizing macrophages to the ingested mitochondria. Collectively, these studies mechanistically link mitophagy and MSC survival with macrophage function, thereby providing a physiologically relevant context for the innate immunomodulatory activity of MSCs.