Generation of a comprehensive molecular cell atlas of the healthy canine lung

peer reviewedSingle cell RNA sequencing (scRNA-seq) is a powerful transcriptomic technique to analyse cell expression profiles across various tissues or conditions, and to identify new cell subpopulations. It has been extensively used in human and mouse studies, and more recently for identification of cellular subpopulations in the bronchoalveolar lavage fluid of dogs. To date, the molecular state of all cell types in canine lung tissue has not been profiled. Such study will help to determine specific cell markers, often lacking in the canine species, and will also provide the foundation for further comparisons with specific lung diseases at single-cell level, such as canine pulmonary fibrosis or neoplasia. In this context, we had a particular interest in fibroblast subpopulations and their expression profiles. Indeed, molecules expressed by fibroblasts (and by cancer-associated fibroblasts) are of potential interest for further development of early markers of disease and of novel molecular fibroblast-targeting therapies. We performed droplet-based scRNA-seq on fresh healthy lung biopsies from three dogs. Two biopsies were collected from dogs euthanized for unrelated reasons, and one was collected from the tumour-free area of a lung lobe resected for primary adenocarcinoma. Biopsies were systematically collected at the peripheral part of the right caudal lobe. Tissues were dissociated to obtain single-cell suspensions, which were loaded into the Chromium Controller (10x Genomics). Clustering, visualization and gene profiling was achieved using the Seurat package in R. Distinct cell populations were identified based on canonical or literature-described cell markers. A total of 22,424 cells were sequenced. Four main cell compartments were identified and individually investigated: epithelial cells (EPCAM+, 5 subpopulations), immune cells (PTPRC+, 17 subpopulations), endothelial cells (PECAM1+, 5 subpopulations) and mesenchymal cells (EPCAM-/PTPRC-/PECAM1-, 10 subpopulations). Clustering resolution was high enough to consistently discriminate different cell subpopulations within classical cell types such as fibroblasts, smooth muscle cells, lymphocytes or macrophages, for example. Among fibroblasts, cluster analysis highlighted subpopulations already identified in humans such as alveolar or adventitial fibroblasts. Differential gene expression profiles were defined for all 37 cell subpopulations, thus identifying new specific cell markers for all cells of the canine lung. This is the first report of scRNA-seq analysis of canine lung tissue, expanding our knowledge of canine distal lung cell subpopulations. This study provides the foundation for comparisons with specific lung diseases at single-cell level, such as canine pulmonary fibrosis or canine pulmonary neoplasia, for which development of emerging therapies are cruelly required

Variations in facial conformation are associated with differences in nasal microbiota in healthy dogs.

peer reviewedBackground: Extrinsic and intrinsic factors have been shown to infuence nasal microbiota (NM) in humans. Very few studies investigated the association between nasal microbiota and factors such as facial/body conformation, age, and environment in dogs. The objectives are to investigate variations in NM in healthy dogs with diferent facial and body conformations. A total of 46 dogs of diferent age, living environment and from 3 diferent breed groups were recruited: 22 meso−/dolichocephalic medium to large breed dogs, 12 brachycephalic dogs and 12 terrier breeds. The nasal bacterial microbiota was assessed through sequencing of 16S rRNA gene (V1-V3 regions) amplicons. Results: We showed major diferences in the NM composition together with increased richness and α-diversity in brachycephalic dogs, compared to meso−/dolichocephalic medium to large dogs and dogs from terrier breeds. Conclusion: Healthy brachycephalic breeds and their unique facial conformation is associated with a distinct NM profle. Description of the NM in healthy dogs serves as a foundation for future researches assessing the changes associated with disease and the modulation of NM communities as a potential treatment

Assessment of SPP1 and FN1 in serum, BALF and lung tissue samples from dogs affected with CIPF

Background: Canine idiopathic pulmonary fibrosis (CIPF) is a chronic disease affecting West Highland white terriers (WHWTs)1,2. Osteopontin (SPP1) and fibronectin (FN1) are associated with pulmonary fibrosis in men3-6 and are overexpressed in bronchoalveolar lavage fluid (BALF) macrophage clusters in CIPF7. Study premise: The aim is to investigate whether these molecules are potential disease markers. SPP1 and FN1 serum and BALF concentrations were measured using canine ELISA kits in CIPF WHWTs (n=24), healthy aged-matched WHWTs (n=13) and healthy terriers (n=15). Proteins were also localized in lung tissue by immunohistochemistry. Results: SPP1 serum concentrations were higher in CIPF compared with healthy WHWTs and terriers, and in healthy WHWTs compared with terriers. There were negatively correlated with PaO2 in WHWTs. Higher SPP1 BALF concentrations were found in CIPF and healthy WHWTs compared with terriers. Intense labelling was reported in all groups in ciliated epithelial cells, smooth muscular cells surrounding large vessels and some macrophages. Moreover, in all CIPF WHWTs, the pneumocytes II and the extra cellular matrix were labelled, while it was the case in only 57% of healthy WHWTs and not present in terriers. FN1 serum concentrations were lower in CIPF and healthy WHWTs compared with terriers. No difference was found between groups in BALF. There was no evidence of differences in FN1 labelling. Conclusions: The results suggest that SPP1 is involved in CIPF pathogenesis and could predispose that breed to the disease. However, further studies are required to determine its interest as biomarker or potential therapeutic target

Expression of Fibroblast Activation Protein in lungs of dogs with idiopathic pulmonary fibrosis and dogs with lung cancer

peer reviewedCanine Idiopathic Pulmonary Fibrosis (CIPF) is a progressive fibrotic interstitial lung disease of unknown aetiology, affecting predominantly the West Highland White Terrier (WHWT) breed. Currently, there is no curative treatment option available. Fibroblast Activation Protein (FAP) is a cell surface protease usually absent from normal tissue but specifically expressed in areas of active tissue remodelling such as in fibroblast foci in human idiopathic pulmonary fibrosis. In humans, it is also upregulated in various types of cancers, either in cancer-associated fibroblasts (CAFs), in cancer cells or in both, depending on the tumour type. The aim of this study was to assess the expression and localization of FAP in the lungs of WHWTs affected with CIPF, in comparison with WHWTs with healthy lungs and dogs with lung cancer. Post-mortem formalin-fixed lung biopsies prepared from WHWTs with CIPF (n=17, age from 10 to 15y), control WHWTs exempt from lung disease (n=4, age from 11 to 15y) and dogs from various breeds with lung cancer (n=8, age from 8 to 14y) were retrospectively used. Included lung neoplasia were adenocarcinomas (n=6), histiocytic sarcoma (n=1) and metastasized mammary adenocarcinoma (n=1). Immunohistochemistry (IHC) was performed using a rabbit anti-human FAP monoclonal antibody (#ab207178). An IHC staining index (absent, low, moderate or high) was attributed according to the percentage of positive cells combined with the staining intensity. FAP was identified in the lungs of 16 out of 17 (94%) WHWTs with CIPF (IHC index high, moderate, or low in respectively 10, 4 and 2 dogs), 2 out of 4 (50%) WHWTs with healthy lungs (1 of each moderate and low), and 7 out of 8 (88%) dogs with lung cancer (high and moderate in respectively 6 and 1 dogs). FAP was expressed by fibroblasts in areas of active fibrosis in CIPF and by CAFs (all types of cancer) and cancer cells (adenocarcinomas only, n=5) in lung tumours. Results of this study showed that FAP is moderately to markedly expressed by fibroblasts in most dogs affected with either CIPF or lung cancer. Accordingly, FAP should be considered as an interesting potential therapeutic target for both diseases and should encourage further studies in the future. The expression of FAP in healthy lungs of WHWTs should be further investigated, particularly in comparison with FAP expression in dogs from other breeds, as it might serve as an indicator of early fibrosis

Antimicrobial discontinuation in dogs with acute aspiration pneumonia based on clinical improvement and normalization of C-reactive protein concentration.

peer reviewed[en] BACKGROUND: Evidence regarding optimal treatment duration in dogs with aspiration pneumonia (AP) and the role of thoracic radiographs (TXR) and lung ultrasonography (LUS) in the long-term follow-up of affected dogs is lacking. C-reactive protein (CRP) is a reliable acute phase protein to monitor bacterial pneumonia in dogs. HYPOTHESIS: Investigate the safety of antimicrobial discontinuation based on clinical improvement and serum CRP normalization, as well as the usefulness of TXR and LUS for follow-up. ANIMALS: Dogs diagnosed with AP and treated with antimicrobials. METHODS: Prospective observational study. Antimicrobials were discontinued based on clinical improvement and serum CRP normalization after 1, 3, or 5 weeks. At each consultation, a quality-of-life questionnaire, physical examination, serum CRP, TXR, and LUS were assessed. Short- (2 weeks) and long-term (>1 month) follow-ups after treatment discontinuation were performed to monitor for possible relapses. RESULTS: Seventeen dogs were included. Antimicrobials were discontinued after 1 week in 12 dogs (70.6%) and 3 weeks in the remaining 5 dogs (29.4%). Short-term relapse was not observed in any dog and long-term relapse was diagnosed in 3 dogs. Thoracic radiographs and LUS were useful for diagnosis, but did not add additional information during follow-up, because image normalization lagged behind clinical improvement and serum CRP normalization. CONCLUSION AND CLINICAL IMPORTANCE: Dogs with AP can be safely and effectively treated using a short-term antimicrobial regimen discontinued after clinical improvement and serum CRP normalization. Imaging might still be useful for complicated cases with a less favorable response to treatment

Evaluation of VEGF-A and CCL2 in dogs with brachycephalic obstructive airway syndrome or canine idiopathic pulmonary fibrosis and in normocephalic dogs

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Response to letter regarding "Comparison of lung ultrasound, chest radiographs, C-reactive protein, and clinical findings in dogs treated for aspiration pneumonia".

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Comparison of lung ultrasound, chest radiographs, C-reactive protein, and clinical findings in dogs treated for aspiration pneumonia.

peer reviewed[en] BACKGROUND: Comparison of clinical findings, chest radiographs (CXR), lung ultrasound (LUS) findings, and C-reactive protein (CRP) concentrations at admission and serial follow-up in dogs with aspiration pneumonia (AP) is lacking. HYPOTHESIS: Lung ultrasound lesions in dogs with AP are similar to those described in humans with community-acquired pneumonia (comAP); the severity of CXR and LUS lesions are similar; normalization of CRP concentration precedes resolution of imaging abnormalities and more closely reflects the clinical improvement of dogs. ANIMALS: Seventeen dogs with AP. METHODS: Prospective observational study. Clinical examination, CXR, LUS, and CRP measurements performed at admission (n = 17), 2 weeks (n = 13), and 1 month after diagnosis (n = 6). All dogs received antimicrobial therapy. Lung ultrasound and CXR canine aspiration scoring systems used to compare abnormalities. RESULTS: B-lines and shred signs with or without bronchograms were identified on LUS in 14 of 17 and 16 of 17, at admission. Chest radiographs and LUS scores differed significantly using both canine AP scoring systems at each time point (18 regions per dog, P < .001). Clinical and CRP normalization occurred in all dogs during follow up. Shred signs disappeared on LUS in all but 1 of 6 dogs at 1 month follow-up, while B-lines and CXR abnormalities persisted in 4 of 6 and all dogs, respectively. CONCLUSION AND CLINICAL IMPORTANCE: Lung ultrasound findings resemble those of humans with comAP and differ from CXR findings. Shred signs and high CRP concentrations better reflect clinical findings during serial evaluation of dogs

Fibroblast activation protein is a cellular marker of fibrotic activity in canine idiopathic pulmonary fibrosis

peer reviewedCanine idiopathic pulmonary fibrosis (CIPF) is a progressive fibrotic interstitial lung disease of unknown etiology, afflicting aging West Highland white terriers (WHWTs) and leading to progressive respiratory failure. Fibroblast activation protein (FAP), a protease overexpressed in many cancers, is upregulated in idiopathic pulmonary fibrosis in humans. The aim of this study was to investigate FAP as a marker of active fibrosis in lung biopsies from WHWTs affected with CIPF, as well as the potential of plasmatic FAP as a biomarker. After establishing a scoring system to evaluate the severity and activity of fibrosis on histopathological lung sections, anti-FAP immunohistochemistry was performed on healthy and CIPF samples. FAP expression was characterized using both visual and digital quantitative pathology software analyses and then correlated to fibrosis severity and activity. Levels of plasmatic FAP in WHWTs affected with CIPF were measured by enzyme-linked immunosorbent assay and compared with healthy dogs. Lung samples from 22 WHWTs affected with CIPF were collected. According to the fibrosis scoring system, they were classified as cases of mild (5), moderate (9) and severe (8) fibrosis and were attributed scores of fibrosis activity. Fifteen healthy lung samples were classified as non-fibrotic. Healthy lung samples were FAP-negative, whereas fibroblasts were FAP-positive in 20 CIPF samples. FAP immunohistochemical expression correlated mildly with fibrosis severity (p < 0.05; R 2 = 0.22) but highly with fibrosis activity scores (p < 0.001; R 2 = 0.68). Digital image analysis detected a higher percentage of FAP-positive cells in areas of active fibrosis (p < 0.001) and FAPpositive cells were distributed outside mature fibrosis lesions, clustered in active fibrosis areas or scattered within alveolar septa. On the other hand, plasmatic FAP was significantly lower in dogs affected with CIPF compared with healthy dogs (p < 0.01). In conclusion, this study provides a valuable histological scoring system to assess the severity and activity of fibrosis in CIPF. It demonstrates that FAP is a good cellular marker of fibrotic activity in CIPF, and thus constitutes a promising target to be exploited for diagnostic and therapeutic applications. Additionally, it suggests that plasmatic FAP, although non-specific, could be altered in CIPF

Utility of Computed Tomographic Angiography for Pulmonary Hypertension Assessment in a Cohort of West Highland White Terriers With or Without Canine Idiopathic Pulmonary Fibrosis

West Highland white terriers (WHWTs) affected with canine idiopathic pulmonary fibrosis (CIPF) are at risk of developing precapillary pulmonary hypertension (PH). In humans, thoracic computed tomography angiography (CTA) is commonly used to diagnose and monitor patients with lower airway diseases. In such patients, CTA helps to identify comorbidities, such as PH, that could negatively impact prognosis. Diameter of the pulmonary trunk (PT), pulmonary trunk-to-aorta ratio (PT/Ao), and right ventricle-to-left ventricle ratio (RV/LV) are CTA parameters commonly used to assess the presence of PH. Pulmonary vein-to-right pulmonary artery ratio (PV/PA) is a new echocardiographic parameter that can be used in dogs to diagnose PH. The primary aim of this study was to evaluate the use of various CTA parameters to diagnose PH. An additional aim was to evaluate the correlation of RV/LV measurements between different CTA planes. CTA and echocardiography were prospectively performed on a total of 47 WHWTs; 22 affected with CIPF and 25 presumed healthy control dogs. Dogs were considered to have PH if pulmonary vein-to-right pulmonary artery ratio (PV/PA) measured on 2D-mode echocardiography was less than to 0.7. WHWTs affected with CIPF had higher PT/Ao compared with control patients. In WHWTs affected with CIPF, PT size was larger in dogs with PH (15.4 mm) compared with dogs without PH (13 mm, p = 0.003). A cutoff value of 13.8 mm predicted PH in WHWTs affected with CIPF with a sensitivity of 90% and a specificity of 87% (AUC = 0.93). High correlations were observed between the different CTA planes of RV/LV. Results suggest that diameter of the PT measured by CTA can be used to diagnose PH in WHWTs with CIPF