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Abstract Background Mediastinal lymph node enlargement is prevalent in patients with idiopathic pulmonary fibrosis (IPF). Studies investigating whether this phenomenon reflects specific immunologic activation are lacking. Methods Programmed cell death-1 (PD-1)/ programmed cell death ligand-1 (PD-L1) expression in mediastinal lymph nodes and lung tissues was analyzed. PD-1, PD-L1 mRNA expression was measured in tracheobronchial lymph nodes of mice following bleomycin-induced injury on day 14. Finally, the effect of the PD-1 inhibitor, pembrolizumab, in bleomycin-induced pulmonary fibrosis was investigated. Results We analyzed mediastinal lymph nodes of thirty-three patients (n = 33, IPF: n = 14, lung cancer: n = 10, concomitant IPF and lung cancer: n = 9) and lung tissues of two hundred nineteen patients (n = 219, IPF: 123, controls: 96). PD-1 expression was increased, while PD-L1 expression was decreased, in mediastinal lymph nodes of patients with IPF compared to lung cancer and in IPF lungs compared to control lungs. Tracheobronchial lymph nodes isolated on day 14 from bleomycin-treated mice exhibited increased size and higher PD-1, PD-L1 mRNA levels compared to saline-treated animals. Pembrolizumab blunted bleomycin-induced lung fibrosis, as indicated by reduction in Ashcroft score and improvement in respiratory mechanics. Conclusions Mediastinal lymph nodes of patients with IPF exhibit differential expression profiles than those of patients with lung cancer indicating distinct immune-mediated pathways regulating fibrogenesis and carcinogenesis. PD-1 expression in mediastinal lymph nodes is in line with lung tissue expression. Lower doses of pembrolizumab might exert antifibrotic effects. Clinical trials aiming to endotype patients based on mediastinal lymph node profiling and accordingly implement targeted therapies such as PD-1 inhibitors are greatly anticipated.

Abstract The activation and accumulation of lung fibroblasts resulting in aberrant deposition of extracellular matrix components, is a pathogenic hallmark of Idiopathic Pulmonary Fibrosis, a lethal and incurable disease. In this report, increased expression of TKS5, a scaffold protein essential for the formation of podosomes, was detected in the lung tissue of Idiopathic Pulmonary Fibrosis patients and bleomycin-treated mice. Τhe profibrotic milieu is found to induce TKS5 expression and the formation of prominent podosome rosettes in lung fibroblasts, that are retained ex vivo, culminating in increased extracellular matrix invasion. Tks5 +/- mice are found resistant to bleomycin-induced pulmonary fibrosis, largely attributed to diminished podosome formation in fibroblasts and decreased extracellular matrix invasion. As computationally predicted, inhibition of src kinase is shown to potently attenuate podosome formation in lung fibroblasts and extracellular matrix invasion, and bleomycin-induced pulmonary fibrosis, suggesting pharmacological targeting of podosomes as a very promising therapeutic option in pulmonary fibrosis.

IntroductionIdiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive interstitial lung disease with dismal prognosis. The underlying pathogenic mechanisms are poorly understood, resulting in a lack of effective treatments. However, recurrent epithelial damage is considered critical for disease initiation and perpetuation, via the secretion of soluble factors that amplify inflammation and lead to fibroblast activation and exuberant deposition of ECM components. Lipocalin-2 (LCN2) is a neutrophil gelatinase-associated lipocalin (NGAL) that has been suggested as a biomarker of kidney damage. LCN2 has been reported to modulate innate immunity, including the recruitment of neutrophils, and to protect against bacterial infections by sequestering iron.MethodsIn silico analysis of publicly available transcriptomic datasets; ELISAs on human IPF patients' bronchoalveolar lavage fluids (BALFs); bleomycin (BLM)-induced pulmonary inflammation and fibrosis and LPS-induced acute lung injury (ALI) in mice: pulmonary function tests, histology, Q-RT-PCR, western blot, and FACS analysis.Results and discussionIncreased LCN2 mRNA expression was detected in the lung tissue of IPF patients negatively correlating with respiratory functions, as also shown for BALF LCN2 protein levels in a cohort of IPF patients. Increased Lcn2 expression was also detected upon BLM-induced pulmonary inflammation and fibrosis, especially at the acute phase correlating with neutrophilic infiltration, as well as upon LPS-induced ALI, an animal model characterized by neutrophilic infiltration. Surprisingly, and non withstanding the limitations of the study and the observed trends, Lcn2−/− mice were found to still develop BLM- or LPS-induced pulmonary inflammation and fibrosis, thus questioning a major pathogenic role for Lcn2 in mice. However, LCN2 qualifies as a surrogate biomarker of pulmonary inflammation and a possible indicator of compromised pulmonary functions, urging for larger studies.

# Background The coronavirus disease 2019 (COVID-19) misinformation and inadequate access to hygiene and sanitation amenities could hamper efforts to contain COVID-19 spread in resource-limited settings. In this study, we describe knowledge of COVID-19 symptoms and preventive measures, sources of information, and access to adequate handwashing among patients with chronic diseases in three Rwandan rural districts during the onset of COVID-19 in Rwanda. # Methods This was a cross-sectional survey conducted among patients who were enrolled in the HIV/AIDS, non-communicable diseases, mental health, oncology, and pediatric development programs at health facilities in Kayonza, Kirehe and Burera districts. The study sample was randomly selected and stratified by district and clinical program. Telephone-based data collection occurred between 23 April and 11 May 2020. Primary caregivers responded to the survey when the selected patient was a child under age 18 or severely ill. We defined good knowledge of COVID-19 symptoms and preventive measures as knowing that a dry cough and fever were common symptoms and social distancing or staying home and regular handwashing could prevent COVID-19 infection. Access to adequate handwashing was defined as living in a household with a handwashing station and regular access to clean water and soap. We used Fisher's exact tests and logistic regression to measure associations between the source of information and good knowledge about COVID-19 and between socio-economic characteristics and access to adequate handwashing. # Results In total, 150 patients and 70 caregivers responded to the survey. Forty-eight (22.3%) respondents had no formal education. Sources of COVID-19 information included mass media (86.8%), local government leaders (27.3%), healthcare workers (15.9%) and social media (6.8%). Twenty-seven percent (n=59) of respondents had good knowledge of COVID-19 symptoms and preventive measures. In the adjusted analysis, getting information from news media was associated with having good knowledge about COVID-19 (adjusted odds ratio, aOR: 5.46; 95% CI: 1.43-20.75]. Seventy-nine (35.9%) respondents reported access to adequate handwashing at home, with access varying significantly by the district in favour of Kayonza (61.3%). # Conclusions COVID-19-related knowledge and access to adequate handwashing were low among patients with chronic diseases at the beginning of the pandemic in Rwanda. Efforts to mitigate COVID-19 spread among chronic care populations may include investment in targeted COVID-19-related education and access to adequate handwashing.

Background: We have previously shown that SHP2 downregulation may predispose fibroblasts to differentiate into myofibroblasts and proposed a role for SHP2 downregulation in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Recent data have shown that SHP2 localizes to the mitochondrial intercristae, and its overexpression enhances mitochondrial metabolism leading to oxidative stress and senescence. Objective: To determine the effect of SHP2 on fibrotic responses. Methods and Results: Primary mouse lung fibroblasts derived from mice carrying a conditional knock-in mutation (D61G/+), rendering the SHP2 catalytic domain constitutively active, had reduced proliferation (1.6-fold, p < 0.05), migration (2-fold, p < 0.05), as well as reduced responsiveness of TGFB-1 induced fibroblasts-to-myofibroblasts differentiation, compared to wild-type ones. Electron microscope analysis revealed that SHP2 D61G/+ mouse lung fibroblasts were characterized by mitochondrial abnormalities, including swollen mitochondria with disrupted electron-lucent cristae and an increased number of autophagosomes compared to wild-type ones. SHP2 D61G/+ MLFs exhibited increased protein levels of autophagy markers, including LC3B-II and p-62, evidence that was confirmed by immunofluorescence analysis. Mitochondrial function analysis revealed that stable (genotype D61G/+) overexpression of SHP2 led to impaired mitochondrial function, as assessed by decreased mitochondrial membrane potential (1.29-fold, p < 0.05), coupling efficiency (1.82 fold, p < 0.05), oxygen consumption rate (1.9-fold, p < 0.05), and increased reactive oxygen species production both at baseline (1.75-fold, p < 0.05) and following H2O2 stimulation (1.63-fold, p < 0.05) compared to wild-type ones (SHP2+/+). SHP2 D61G/+ mouse lung fibroblasts showed enhanced AMPK activity, as well as decreased activation of the mTORC1 signaling pathway, potentially leading to ineffective mitochondrial metabolism and increased autophagy. Conclusions: SHP2 attenuates fibrotic responses in fibroblast cell lines through negative regulation of mitochondrial metabolism and induction of autophagy. SHP2 activation may represent a promising therapeutic strategy for patients with fibrotic lung diseases.

Within the Interstitial Lung Diseases (ILD), patients with idiopathic pulmonary fibrosis (IPF) and a subset of those with non-IPF fibrotic ILD have a distinct clinical phenotype of progression despite management. This group of patients has been collectively termed the progressive fibrotic phenotype (PFP). Their early recognition may facilitate access to antifibrotic therapies to prevent or slow progression. Macrophages/monocytes within the lung orchestrate the progression and maintenance of fibrosis. A novel role for monocyte-derived macrophages during tissue damage and wound healing is the expression of collagens. We examined Collagen 1a1 expression in airway macrophages from ILD patients at diagnosis. COL1A1 mRNA levels from BAL cells were elevated in IPF and Non-IPF patients. The presence of a UIP pattern and a subsequent progressive phenotype were significantly associated with the higher BAL COL1A1 levels. In Non-IPF patients, higher COL1A1 levels were associated with a more than twofold increase in mortality. The intracellular localisation of COL1A1 in airway macrophages was demonstrated by confocal microscopy in CD45 and CD163 co-staining assays. Additionally, airway macrophages co-expressed COL1A1 with the profibrotic SPP1 gene product osteopontin. The levels of SPP1 mRNA and OPN in the BAL were significantly higher in IPF and Non-IPF patients relative to healthy. Our results suggest that profibrotic airway macrophages are increased in the BAL of patients with IPF and other ILDs and co-express COL1A1 and OPN. Importantly, COL1A1 expression by pro-fibrotic airway macrophages could be a marker of disease progression and poor survival in ILDs.

The microbiome is emerging as a major player in tissue homeostasis in health and disease. Gut microbiome dysbiosis correlates with several autoimmune and metabolic diseases, while high-fat diets and ensuing obesity are known to affect the complexity and diversity of the microbiome, thus modulating pathophysiology. Moreover, the existence of a gut-liver microbial axis has been proposed, which may extend to the lung. In this context, we systematically compared the microbiomes of the gut, liver, and lung of mice fed a high-fat diet to those of littermates fed a matched control diet. We carried out deep sequencing of seven hypervariable regions of the 16S rRNA microbial gene to examine microbial diversity in the tissues of interest. Comparison of the local microbiomes indicated that lung tissue has the least diverse microbiome under healthy conditions, while microbial diversity in the healthy liver clustered closer to the gut. Obesity increased microbial complexity in all three tissues, with lung microbial diversity being the most modified. Obesity promoted the expansion of Firmicutes along the gut-liver-lung axis, highlighting staphylococcus as a possible pathologic link between obesity and systemic pathophysiology, especially in the lungs.

Phenolic acids represent abundant components contained in human diet. However, the negative charge in their carboxylic group limits their capacity to diffuse through biological membranes, thus hindering their access to cell interior. In order to promote the diffusion of rosmarinic acid through biological membranes, we synthesized several lipophilic ester- and amide-derivatives of this compound and evaluated their capacity to prevent H2O2-induced DNA damage and apoptosis in cultured human cells. Esterification of the carboxylic moiety with lipophilic groups strongly enhanced the capacity of rosmarinic acid to protect cells. On the other hand, the amide-derivatives were somewhat less effective but exerted less cytotoxicity at high concentrations. Cell uptake experiments, using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), illustrated different levels of intracellular accumulation among the ester- and amide-derivatives, with the first being more effectively accumulated, probably due to their extensive hydrolysis inside the cells. In conclusion, these results highlight the hitherto unrecognized fundamental importance of derivatization of diet-derived phenolic acids to unveil their biological potential.

Hereditary Hemorrhagic Telangiectasia type 1 (HHT1) is an autosomal dominant inherited disease characterized by arteriovenous malformations and hemorrhage. HHT1 is caused by mutations in ENDOGLIN, which encodes an ancillary receptor for Transforming Growth Factor-β/Bone Morphogenetic Protein-9 expressed in all vascular endothelial cells. Haploinsufficiency is widely accepted as the underlying mechanism for HHT1. However, it remains intriguing that only some, but not all, vascular beds are affected, as these causal gene mutations are present in vasculature throughout the body. Here, we have examined the endoglin expression levels in the blood vessels of multiple organs in mice and in humans. We found a positive correlation between low basal levels of endoglin and the general prevalence of clinical manifestations in selected organs. Endoglin was found to be particularly low in the skin, the earliest site of vascular lesions in HHT1, and even undetectable in the arteries and capillaries of heterozygous endoglin mice. Endoglin levels did not appear to be associated with organ-specific vascular functions. Instead, our data revealed a critical endoglin threshold compatible with the haploinsufficiency model, below which endothelial cells independent of their tissue of origin exhibited abnormal responses to Vascular Endothelial Growth Factor. Our results support the development of drugs promoting endoglin expression as potentially protective.

Although it is known that Mediterranean diet plays an important role in maintaining human health, the underlying molecular mechanisms remain largely unknown. The aim of this investigation was to elucidate the potential role of ortho-dihydroxy group containing natural compounds in H2O2-induced DNA damage and apoptosis. For this purpose, the main phenolic alcohols of olive oil, namely hydroxytyrosol and tyrosol, were examined for their ability to protect cultured cells under conditions of oxidative stress. A strong correlation was observed between the ability of hydroxytyrosol to mitigate intracellular labile iron level and the protection offered against H2O2-induced DNA damage and apoptosis. On the other hand, tyrosol, which lacks the ortho-dihydroxy group, was ineffective. Moreover, hydroxytyrosol (but not tyrosol), was able to diminish the late sustained phase of H2O2-induced JNK and p38 phosphorylation. The derangement of intracellular iron homeostasis, following exposure of cells to H2O2, played pivotal role both in the induction of DNA damage and the initiation of apoptotic signaling. The presented results suggest that the protective effects exerted by ortho-dihydroxy group containing dietary compounds against oxidative stress-induced cell damage are linked to their ability to influence changes in the intracellular labile iron homeostasis.

One of the prevailing perceptions regarding the ageing of cells and organisms is the intracellular gradual accumulation of oxidatively damaged macromolecules, leading to the decline of cell and organ function (free radical theory of ageing). This chemically undefined material known as “lipofuscin,” “ceroid,” or “age pigment” is mainly formed through unregulated and nonspecific oxidative modifications of cellular macromolecules that are induced by highly reactive free radicals. A necessary precondition for reactive free radical generation and lipofuscin formation is the intracellular availability of ferrous iron (Fe2+) (“labile iron”), catalyzing the conversion of weak oxidants such as peroxides, to extremely reactive ones like hydroxyl (HO•) or alcoxyl (RO•) radicals. If the oxidized materials remain unrepaired for extended periods of time, they can be further oxidized to generate ultimate over-oxidized products that are unable to be repaired, degraded, or exocytosed by the relevant cellular systems. Additionally, over-oxidized materials might inactivate cellular protection and repair mechanisms, thus allowing for futile cycles of increasingly rapid lipofuscin accumulation. In this review paper, we present evidence that the modulation of the labile iron pool distribution by nutritional or pharmacological means represents a hitherto unappreciated target for hampering lipofuscin accumulation and cellular ageing.