Inhalable Textile Microplastic Fibers Impair Airway Epithelial Differentiation.

Rationale: Microplastics are a pressing global concern, and inhalation of microplastic fibers has been associated with interstitial and bronchial inflammation in flock workers. However, how microplastic fibers affect the lungs is unknown. Objectives: Our aim was to assess the effects of 12 × 31 µm nylon 6,6 (nylon) and 15 × 52 µm polyethylene terephthalate (polyester) textile microplastic fibers on lung epithelial growth and differentiation. Methods: We used human and murine alveolar and airway-type organoids as well as air-liquid interface cultures derived from primary lung epithelial progenitor cells and incubated these with either nylon or polyester fibers or nylon leachate. In addition, mice received one dose of nylon fibers or nylon leachate, and, 7 days later, organoid-forming capacity of isolated epithelial cells was investigated. Measurements and Main Results: We observed that nylon microfibers, more than polyester, inhibited developing airway organoids and not established ones. This effect was mediated by components leaching from nylon. Epithelial cells isolated from mice exposed to nylon fibers or leachate also formed fewer airway organoids, suggesting long-lasting effects of nylon components on epithelial cells. Part of these effects was recapitulated in human air-liquid interface cultures. Transcriptomic analysis revealed upregulation of Hoxa5 after exposure to nylon fibers. Inhibiting Hoxa5 during nylon exposure restored airway organoid formation, confirming Hoxa5's pivotal role in the effects of nylon. Conclusions: These results suggest that components leaching from nylon 6,6 may especially harm developing airways and/or airways undergoing repair, and we strongly encourage characterization in more detail of both the hazard of and the exposure to microplastic fibers.

The lung extracellular matrix protein landscape in severe early-onset and moderate chronic obstructive pulmonary disease.

Extracellular matrix (ECM) remodeling has been implicated in the irreversible obstruction of airways and destruction of alveolar tissue in chronic obstructive pulmonary disease (COPD). Studies investigating differences in the lung ECM in COPD have mainly focused on some collagens and elastin, leaving an array of ECM components unexplored. We investigated the differences in the ECM landscape comparing severe-early onset (SEO-) COPD and moderate COPD to control lung tissue for collagen type I α chain 1 (COL1A1), COL6A1, COL6A2, COL14A1, fibulin 2 and 5 (FBLN2, FBLN5), latent transforming growth factor-beta binding protein 4 (LTBP4), lumican (LUM), versican (VCAN), decorin (DCN), and elastin (ELN) using image analysis and statistical modelling. Percentage area and/or mean intensity of expression of LUM in the parenchyma, and COL1A1, FBLN2, LTBP4, DCN, and VCAN in the airway walls, was proportionally lower in COPD compared to controls. Lowered levels of most ECM proteins were associated with decreasing FEV1 measurements, indicating a relationship with disease severity. Furthermore, we identified six unique ECM signatures where LUM and COL6A1 in parenchyma and COL1A1, FBLN5, DCN, and VCAN in airway walls appear essential in reflecting the presence and severity of COPD. These signatures emphasize the need to examine groups of proteins to represent an overall difference in the ECM landscape in COPD, that are more likely to be related to functional effects, than individual proteins. Our study revealed differences in the lung ECM landscape between control and COPD and between SEO and moderate COPD signifying distinct pathological processes in the different subgroups.

Osteoprotegerin is an Early Marker of the Fibrotic Process and of Antifibrotic Treatment Responses in Ex Vivo Lung Fibrosis.

BACKGROUND: Lung fibrosis is a chronic lung disease with a high mortality rate with only two approved drugs (pirfenidone and nintedanib) to attenuate its progression. To date, there are no reliable biomarkers to assess fibrosis development and/or treatment effects for these two drugs. Osteoprotegerin (OPG) is used as a serum marker to diagnose liver fibrosis and we have previously shown it associates with lung fibrosis as well. METHODS: Here we used murine and human precision-cut lung slices to investigate the regulation of OPG in lung tissue to elucidate whether it tracks with (early) fibrosis development and responds to antifibrotic treatment to assess its potential use as a biomarker. RESULTS: OPG mRNA expression in murine lung slices was higher after treatment with profibrotic cytokines TGFß1 or IL13, and closely correlated with Fn and PAI1 mRNA expression. More OPG protein was released from fibrotic human lung slices than from the control human slices and from TGFß1 and IL13-stimulated murine lung slices compared to control murine slices. This OPG release was inhibited when murine slices were treated with pirfenidone or nintedanib. OPG release from human fibrotic lung slices was inhibited by pirfenidone treatment. CONCLUSION: OPG can already be detected during the early stages of fibrosis development and responds, both in early- and late-stage fibrosis, to treatment with antifibrotic drugs currently on the market for lung fibrosis. Therefore, OPG should be further investigated as a potential biomarker for lung fibrosis and a potential surrogate marker for treatment effect.

Macrophage migration inhibitory factor family proteins are multitasking cytokines in tissue injury.

The family of macrophage migration inhibitory factor (MIF) proteins in humans consist of MIF, its functional homolog D-dopachrome tautomerase (D-DT, also known as MIF-2) and the relatively unknown protein named DDT-like (DDTL). MIF is a pleiotropic cytokine with multiple properties in tissue homeostasis and pathology. MIF was initially found to associate with inflammatory responses and therefore established a reputation as a pro-inflammatory cytokine. However, increasing evidence demonstrates that MIF influences many different intra- and extracellular molecular processes important for the maintenance of cellular homeostasis, such as promotion of cellular survival, antioxidant signaling, and wound repair. In contrast, studies on D-DT are scarce and on DDTL almost nonexistent and their functions remain to be further investigated as it is yet unclear how similar they are compared to MIF. Importantly, the many and sometimes opposing functions of MIF suggest that targeting MIF therapeutically should be considered carefully, taking into account timing and severity of tissue injury. In this review, we focus on the latest discoveries regarding the role of MIF family members in tissue injury, inflammation and repair, and highlight the possibilities of interventions with therapeutics targeting or mimicking MIF family proteins.

Osteoprotegerin Expression in Liver is Induced by IL13 through TGFß.

BACKGROUND/AIMS: Osteoprotegerin (OPG) is a profibrotic mediator produced by myofibro-blasts under influence of transforming growth factor ß (TGFß). Its expression in experimental models of liver fibrosis correlates well with disease severity and treatment responses. The regulation of OPG in liver tissue is largely unknown and we therefore set out to elucidate which growth factors/interleukins associated with fibrosis induce OPG and through which pathways. METHODS: Precision-cut liver slices of wild type and STAT6-deficient mice and 3T3 fibroblasts were used to investigate the effects of TGFß, interleukin (IL) 13 (IL13), IL1ß, and platelet-derived growth factor BB (PDGF-BB) on expression of OPG. OPG protein was measure by ELISA, whereas OPG mRNA and expression of other relevant genes was measured by qPCR. RESULTS: In addition to TGFß, only IL13 and not PDGF-BB or IL1ß could induce OPG expression in 3T3 fibroblasts and liver slices. This IL13-dependent induction was not shown in liver slices of STAT6-deficient mice and when wild type slices were cotreated with TGFß receptor 1 kinase inhibitor galunisertib, STAT6 inhibitor AS1517499, or AP1 inhibitor T5224. This suggests that the OPG-inducing effect of IL13 is mediated through IL13 receptor α1-activation and subsequent STAT6-dependent upregulation of IL13 receptor α2, which in turn activates AP1 and induces production of TGFß and subsequent production of OPG. CONCLUSION: We have shown that IL13 induces OPG release by liver tissue through a TGFß-dependent pathway involving both the α1 and the α2 receptor of IL13 and transcription factors STAT6 and AP1. OPG may therefore be a novel target for the treatment liver fibrosis as it is mechanistically linked to two important regulators of fibrosis in liver, namely IL13 and TGFß1.

4-Iodopyrimidine Labeling Reveals Nuclear Translocation and Nuclease Activity for Both MIF and MIF2.

Macrophage migration inhibitory factor (MIF) and its homolog MIF2 (also known as D-dopachrome tautomerase or DDT) play key roles in cell growth and immune responses. MIF and MIF2 expression is dysregulated in cancers and neurodegenerative diseases. Accurate and convenient detection of MIF and MIF2 will facilitate research on their roles in cancer and other diseases. Herein, we report the development and application of a 4-iodopyrimidine based probe 8 for the selective labeling of MIF and MIF2. Probe 8 incorporates a fluorophore that allows in situ imaging of these two proteins. This enabled visualization of the translocation of MIF2 from the cytoplasm to the nucleus upon methylnitronitrosoguanidine stimulation of HeLa cells. This observation, combined with literature on nuclease activity for MIF, enabled the identification of nuclease activity for MIF2 on human genomic DNA.

Macrophage-stroma interactions in fibrosis: biochemical, biophysical, and cellular perspectives.

Fibrosis results from aberrant wound healing and is characterized by an accumulation of extracellular matrix, impairing the function of an affected organ. Increased deposition of extracellular matrix proteins, disruption of matrix degradation, but also abnormal post-translational modifications alter the biochemical composition and biophysical properties of the tissue microenvironment - the stroma. Macrophages are known to play an important role in wound healing and tissue repair, but the direct influence of fibrotic stroma on macrophage behaviour is still an under-investigated element in the pathogenesis of fibrosis. In this review, the current knowledge on interactions between macrophages and (fibrotic) stroma will be discussed from biochemical, biophysical, and cellular perspectives. Furthermore, we provide future perspectives with regard to how macrophage-stroma interactions can be examined further to ultimately facilitate more specific targeting of these interactions in the treatment of fibrosis. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Combined Metabolic and Chemical (CoMetChem) Labeling Using Stable Isotopes-a Strategy to Reveal Site-Specific Histone Acetylation and Deacetylation Rates by LC-MS.

Histone acetylation is an important, reversible post-translational protein modification and a hallmark of epigenetic regulation. However, little is known about the dynamics of this process, due to the lack of analytical methods that can capture site-specific acetylation and deacetylation reactions. We present a new approach that combines metabolic and chemical labeling (CoMetChem) using uniformly 13C-labeled glucose and stable isotope-labeled acetic anhydride. Thereby, chemically equivalent, fully acetylated histone species are generated, enabling accurate relative quantification of site-specific lysine acetylation dynamics in tryptic peptides using high-resolution mass spectrometry. We show that CoMetChem enables site-specific quantification of the incorporation or loss of lysine acetylation over time, allowing the determination of reaction rates for acetylation and deacetylation. Thus, the CoMetChem methodology provides a comprehensive description of site-specific acetylation dynamics.

Proteolysis Targeting Chimera (PROTAC) for Macrophage Migration Inhibitory Factor (MIF) Has Anti-Proliferative Activity in Lung Cancer Cells.

Macrophage migration inhibitory factor (MIF) is involved in protein-protein interactions that play key roles in inflammation and cancer. Current strategies to develop small molecule modulators of MIF functions are mainly restricted to the MIF tautomerase active site. Here, we use this site to develop proteolysis targeting chimera (PROTAC) in order to eliminate MIF from its protein-protein interaction network. We report the first potent MIF-directed PROTAC, denoted MD13, which induced almost complete MIF degradation at low micromolar concentrations with a DC50 around 100 nM in A549 cells. MD13 suppresses the proliferation of A549 cells, which can be explained by deactivation of the MAPK pathway and subsequent induction of cell cycle arrest at the G2/M phase. MD13 also exhibits antiproliferative effect in a 3D tumor spheroid model. In conclusion, we describe the first MIF-directed PROTAC (MD13) as a research tool, which also demonstrates the potential of PROTACs in cancer therapy.

The role of MIF in chronic lung diseases: looking beyond inflammation.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been associated with many diseases. Most studies found in literature describe MIF as a proinflammatory cytokine involved in chronic inflammatory conditions, but evidence from last years suggests that many of its key effects are not directly related to inflammation. In fact, MIF is constitutively expressed in most human tissues and in some cases in high levels, which does not reflect the pattern of expression of a classic proinflammatory cytokine. Moreover, MIF is highly expressed during embryonic development and decreases during adulthood, which point toward a more likely role as growth factor. Accordingly, MIF knockout mice develop age-related spontaneous emphysema, suggesting that MIF presence (e.g., in younger individuals and wild-type animals) is part of a healthy lung. In view of this new line of evidence, we aimed to review data on the role of MIF in the pathogenesis of chronic lung diseases.

Prenatal smoke exposure dysregulates lung epithelial cell differentiation in mouse offspring: role for AREG-induced EGFR signaling.

Prenatal smoke exposure is a risk factor for impaired lung development in children. Recent studies have indicated that amphiregulin (AREG), which is a ligand of the epidermal growth factor receptor (EGFR), has a regulatory role in airway epithelial cell differentiation. In this study, we investigated the effect of prenatal smoke exposure on lung epithelial cell differentiation and linked this with AREG-EGFR signaling in 1-day-old mouse offspring. Bronchial and alveolar epithelial cell differentiations were assessed by immunohistochemistry. Areg, epidermal growth factor (Egf), and mRNA expressions of specific markers for bronchial and alveolar epithelial cells were assessed by RT-qPCR. The results in neonatal lungs were validated in an AREG-treated three-dimensional mouse lung organoid model. We found that prenatal smoke exposure reduced the number of ciliated cells and the expression of the cilia-related transcription factor Foxj1, whereas it resulted in higher expression of mucus-related transcription factors Spdef and Foxm1 in the lung. Moreover, prenatally smoke-exposed offspring had higher numbers of alveolar epithelial type II cells (AECII) and lower expression of the AECI-related Pdpn and Gramd2 markers. This was accompanied by higher expression of Areg and lower expression of Egf in prenatally smoke-exposed offspring. In bronchial organoids, AREG treatment resulted in fewer ciliated cells and more basal cells when compared with non-treated bronchiolar organoids. In alveolar organoids, AREG treatment led to more AECII cells than non-treated AECII cells. Taken together, the observed impaired bronchial and alveolar cell development in prenatally smoke-exposed neonatal offspring may be induced by increased AREG-EGFR signaling.

Time-Resolved Quantification of Nanoparticle Uptake, Distribution, and Impact in Precision-Cut Liver Slices.

Much effort within the nanosafety field is currently focused on the use of advanced in vitro models to reduce the gap between in vitro and in vivo studies. Within this context, precision-cut tissue slices are a unique ex vivo model to investigate nanoparticle impact using live tissue from laboratory animals and even humans. However, several aspects of the basic mechanisms of nanoparticle interactions with tissue have not yet been elucidated. To this end, liver slices are exposed to carboxylated and amino-modified polystyrene known to have a different impact on cells. As observed in standard cell cultures, amino-modified polystyrene nanoparticles induce apoptosis, and their impact is affected by the corona forming on their surface in biological fluids. Subsequently, a detailed time-resolved study of nanoparticle uptake and distribution in the tissue is performed, combining fluorescence imaging and flow cytometry on cells recovered after tissue digestion. As observed in vivo, the Kupffer cells accumulate high nanoparticle amounts and, interestingly, they move within the tissue towards the slice borders. Similar observations are reproduced in liver slices from human tissue. Thus, tissue slices can be used to reproduce ex vivo important features of nanoparticle outcomes in the liver and study nanoparticle impact on real tissue.

The different faces of the macrophage in asthma.

PURPOSE OF REVIEW: Asthma is a chronic inflammatory disease in which changes in macrophage polarization have been shown to contribute to the pathogenesis. The present review discusses the contribution of changes in macrophage function to asthma related to polarization changes and elaborates on possible therapeutic strategies targeting macrophage function and polarization. RECENT FINDINGS: Macrophage function alterations were shown to contribute to asthma pathology in several ways. One is by impaired phagocytosis and efferocytosis. Another is by changing inflammation, by altered (anti)inflammatory cytokine production and induction of the inflammasome. Finally, macrophages can contribute to remodeling in asthma, although little evidence is present in humans yet.Novel therapeutic strategies targeting macrophages include dampening inflammation by changing polarization or by inhibiting the NLRP3 inflammasome, and by targeting efferocytosis. However, many of these studies were performed in animal models leaving their translation to the clinic for future research. SUMMARY: The present review emphasizes the contribution of altered macrophage function to asthma, gives insight in possible new therapeutic strategies targeting macrophages, and indicates which knowledge gaps remain open.

Oxidative stress and macrophages: driving forces behind exacerbations of asthma and chronic obstructive pulmonary disease?

Oxidative stress is a common feature of obstructive airway diseases like asthma and chronic obstructive pulmonary disease (COPD). Lung macrophages are key innate immune cells that can generate oxidants and are known to display aberrant polarization patterns and defective phagocytic responses in these diseases. Whether these characteristics are linked in one way or another and whether they contribute to the onset and severity of exacerbations in asthma and COPD remain poorly understood. Insight into oxidative stress, macrophages, and their interactions may be important in fully understanding acute worsening of lung disease. This review therefore highlights the current state of the art regarding the role of oxidative stress and macrophages in exacerbations of asthma and COPD. It shows that oxidative stress can attenuate macrophage function, which may result in impaired responses toward exacerbating triggers and may contribute to exaggerated inflammation in the airways.

Enhanced Bruton's tyrosine kinase in B-cells and autoreactive IgA in patients with idiopathic pulmonary fibrosis.

RATIONALE: Idiopathic Pulmonary Fibrosis (IPF) is thought to be triggered by repeated alveolar epithelial cell injury. Current evidence suggests that aberrant immune activation may contribute. However, the role of B-cell activation remains unclear. We determined the phenotype and activation status of B-cell subsets and evaluated the contribution of activated B-cells to the development of lung fibrosis both in humans and in mice. METHODS: B-cells in blood, mediastinal lymph node, and lung single-cell suspensions of IPF patients and healthy controls (HC) were characterized using 14-color flow cytometry. Mice were exposed to bleomycin to provoke pulmonary fibrosis. RESULTS: More IgA+ memory B-cells and plasmablasts were found in blood (n = 27) and lungs (n = 11) of IPF patients compared to HC (n = 21) and control lungs (n = 9). IPF patients had higher levels of autoreactive IgA in plasma, which correlated with an enhanced decline of forced vital capacity (p = 0.002, r = - 0.50). Bruton's tyrosine kinase expression was higher in circulating IPF B-cells compared to HC, indicating enhanced B-cell activation. Bleomycin-exposed mice had increased pulmonary IgA+ germinal center and plasma cell proportions compared to control mice. The degree of lung fibrosis correlated with pulmonary germinal center B-cell proportions (p = 0.010, r = 0.88). CONCLUSION: Our study demonstrates that IPF patients have more circulating activated B-cells and autoreactive IgA, which correlate with disease progression. These B-cell alterations were also observed in the widely used mouse model of experimental pulmonary fibrosis. Autoreactive IgA could be useful as a biomarker for disease progression in IPF.

Sexual maturation protects against development of lung inflammation through estrogen.

Increasing levels of estrogen and progesterone are suggested to play a role in the gender switch in asthma prevalence during puberty. We investigated whether the process of sexual maturation in mice affects the development of lung inflammation in adulthood and the contributing roles of estrogen and progesterone during this process. By inducing ovalbumin-induced lung inflammation in sexually mature and immature (ovariectomized before sexual maturation) adult mice, we showed that sexually immature adult mice developed more eosinophilic lung inflammation. This protective effect of "puberty" appears to be dependent on estrogen, as estrogen supplementation at the time of ovariectomy protected against development of lung inflammation in adulthood whereas progesterone supplementation did not. Investigating the underlying mechanism of estrogen-mediated protection, we found that estrogen-treated mice had higher expression of the anti-inflammatory mediator secretory leukoprotease inhibitor (SLPI) and lower expression of the proasthmatic cytokine IL-33 in parenchymal lung tissue and that their expressions colocalized with type II alveolar epithelial cells (AECII). Treating AECII directly with SLPI significantly inhibited IL-33 production upon stimulation with ATP. Our data suggest that estrogen during puberty has a protective effect on asthma development, which is accompanied by induction of anti-inflammatory SLPI production and inhibition of proinflammatory IL-33 production by AECII.

Chronic exposure to glucocorticoids shapes gene expression and modulates innate and adaptive activation pathways in macrophages with distinct changes in leukocyte attraction.

Glucocorticoids (GCs) have been used for more than 50 y as immunosuppressive drugs, yet their efficacy in macrophage-dominated disorders, such as chronic obstructive pulmonary disease, is debated. Little is known how long-term GC treatment affects macrophage responses in inflammatory conditions. In this study, we compared the transcriptome of human macrophages, matured in the presence or absence of fluticasone propionate (FP), and their ability to initiate or sustain classical activation, mimicked using acute LPS and chronic IFN-γ stimulation, respectively. We identified macrophage gene expression networks, modulated by FP long-term exposure, and specific patterns of IFN-γ- and LPS-induced genes that were resistant, inhibited, or exacerbated by FP. Results suggest that long-term treatment with GCs weakens adaptive immune signature components of IFN-γ and LPS gene profiles by downmodulating MHC class II and costimulatory molecules, but strengthens innate signature components by maintaining and increasing expression of chemokines involved in phagocyte attraction. In a mouse model of chronic obstructive pulmonary disease, GC treatment induced higher chemokine levels, and this correlated with enhanced recruitment of leukocytes. Thus, GCs do not generally suppress macrophage effector functions, but they cause a shift in the innate-adaptive balance of the immune response, with distinct changes in the chemokine-chemokine receptor network.

Distinct macrophage phenotypes in allergic and nonallergic lung inflammation.

Chronic exposure to farm environments is a risk factor for nonallergic lung disease. In contrast to allergic asthma, in which type 2 helper T cell (Th2) activation is dominant, exposure to farm dust extracts (FDE) induces Th1/Th17 lung inflammation, associated with neutrophil infiltration. Macrophage influx is a common feature of both types of lung inflammation, allergic and nonallergic. However, macrophage functions and phenotypes may vary according to their polarized state, which is dependent on the cytokine environment. In this study, we aimed to characterize and quantify the lung macrophage populations in two established murine models of allergic and nonallergic lung inflammation by means of fluorescence-activated cell sorting and immunohistochemistry. We demonstrated that, whereas in allergic asthma M2-dominant macrophages predominated in the lungs, in nonallergic inflammation M1-dominant macrophages were more prevalent. This was confirmed in vitro using a macrophage cell line, where FDE exerted a direct effect on macrophages, inducing M1-dominant polarization. The polarization of macrophages diverged depending on the exposure and inflammatory status of the tissue. Interfering with this polarization could be a target for treatment of different types of lung inflammation.

Genetic programs expressed in resting and IL-4 alternatively activated mouse and human macrophages: similarities and differences.

The molecular repertoire of macrophages in health and disease can provide novel biomarkers for diagnosis, prognosis, and treatment. Th2-IL-4­activated macrophages (M2) have been associated with important diseases in mice, yet no specific markers are available for their detection in human tissues. Although mouse models are widely used for macrophage research, translation to the human can be problematic and the human macrophage system remains poorly described. In the present study, we analyzed and compared the transcriptome and proteome of human and murine macrophages under resting conditions (M0) and after IL-4 activation (M2). We provide a resource for tools enabling macrophage detection in human tissues by identifying a set of 87 macrophage-related genes. Furthermore, we extend current understanding of M2 activation in different species and identify Transglutaminase 2 as a conserved M2 marker that is highly expressed by human macrophages and monocytes in the prototypic Th2 pathology asthma.

Microplastic and plastic pollution: impact on respiratory disease and health.

Throughout their lifecycle, from production to use and upon disposal, plastics release chemicals and particles known as micro- and nanoplastics (MNPs) that can accumulate in the environment. MNPs have been detected in different locations of the human body, including in our lungs. This is likely a consequence of MNP exposure through the air we breathe. Yet, we still lack a comprehensive understanding of the impact that MNP exposure may have on respiratory disease and health. In this review, we have collated the current body of evidence on the implications of MNP inhalation on human lung health from in vitro, in vivo and occupational exposure studies. We focused on interactions between MNP pollution and different specific lung-resident cells and respiratory diseases. We conclude that it is evident that MNPs possess the capacity to affect lung tissue in disease and health. Yet, it remains unclear to which extent this occurs upon exposure to ambient levels of MNPs, emphasising the need for a more comprehensive evaluation of environmental MNP exposure levels in everyday life.