The nature of chronic rejection after lung transplantation: a murine orthotopic lung transplant study.

Introduction: Chronic rejection is a major complication post-transplantation. Within lung transplantation, chronic rejection was considered as airway centred. Chronic Lung Allograft Dysfunction (CLAD), defined to cover all late chronic complications, makes it more difficult to understand chronic rejection from an immunological perspective. This study investigated the true nature, timing and location of chronic rejection as a whole, within mouse lung transplantation. Methods: 40 mice underwent an orthotopic left lung transplantation, were sacrificed at day 70 and evaluated by histology and in vivo µCT. For timing and location of rejection, extra grafts were sacrificed at day 7, 35, 56 and investigated by ex vivo µCT or single cell RNA (scRNA) profiling. Results: Chronic rejection originated as innate inflammation around small arteries evolving toward adaptive organization with subsequent end-arterial fibrosis and obliterans. Subsequently, venous and pleural infiltration appeared, followed by airway related bronchiolar folding and rarely bronchiolitis obliterans was observed. Ex vivo µCT and scRNA profiling validated the time, location and sequence of events with endothelial destruction and activation as primary onset. Conclusion: Against the current belief, chronic rejection in lung transplantation may start as an arterial response, followed by responses in venules, pleura, and, only in the late stage, bronchioles, as may be seen in some but not all patients with CLAD.

A Pathology-based Case Series of Influenza- and COVID-19-associated Pulmonary Aspergillosis: The Proof Is in the Tissue.

Rationale: Invasive pulmonary aspergillosis has emerged as a frequent coinfection in severe coronavirus disease (COVID-19), similarly to influenza, yet the clinical invasiveness is more debated. Objectives: We investigated the invasive nature of pulmonary aspergillosis in histology specimens of influenza and COVID-19 ICU fatalities in a tertiary care center. Methods: In this monocentric, descriptive, retrospective case series, we included adult ICU patients with PCR-proven influenza/COVID-19 respiratory failure who underwent postmortem examination and/or tracheobronchial biopsy during ICU admission from September 2009 until June 2021. Diagnosis of probable/proven viral-associated pulmonary aspergillosis (VAPA) was made based on the Intensive Care Medicine influenza-associated pulmonary aspergillosis and the European Confederation of Medical Mycology (ECMM) and the International Society for Human and Animal Mycology (ISHAM) COVID-19-associated pulmonary aspergillosis consensus criteria. All respiratory tissues were independently reviewed by two experienced pathologists. Measurements and Main Results: In the 44 patients of the autopsy-verified cohort, 6 proven influenza-associated and 6 proven COVID-19-associated pulmonary aspergillosis diagnoses were identified. Fungal disease was identified as a missed diagnosis upon autopsy in 8% of proven cases (n = 1/12), yet it was most frequently found as confirmation of a probable antemortem diagnosis (n = 11/21, 52%) despite receiving antifungal treatment. Bronchoalveolar lavage galactomannan testing showed the highest sensitivity for VAPA diagnosis. Among both viral entities, an impeded fungal growth was the predominant histologic pattern of pulmonary aspergillosis. Fungal tracheobronchitis was histologically indistinguishable in influenza (n = 3) and COVID-19 (n = 3) cases, yet macroscopically more extensive at bronchoscopy in influenza setting. Conclusions: A proven invasive pulmonary aspergillosis diagnosis was found regularly and with a similar histological pattern in influenza and in COVID-19 ICU case fatalities. Our findings highlight an important need for VAPA awareness, with an emphasis on mycological bronchoscopic work-up.

Visualising SARS-CoV-2 infection of the lung in deceased COVID-19 patients.

BACKGROUND: SARS-CoV-2 is a single-stranded positive-sense RNA virus. Several negative-sense SARS-CoV-2 RNA species, both full-length genomic and subgenomic, are produced transiently during viral replication. Methodologies for rigorously characterising cell tropism and visualising ongoing viral replication at single-cell resolution in histological sections are needed to assess the virological and pathological phenotypes of future SARS-CoV-2 variants. We aimed to provide a robust methodology for examining the human lung, the major target organ of this RNA virus. METHODS: A prospective cohort study took place at the University Hospitals Leuven in Leuven, Belgium. Lung samples were procured postmortem from 22 patients who died from or with COVID-19. Tissue sections were fluorescently stained with the ultrasensitive single-molecule RNA in situ hybridisation platform of RNAscope combined with immunohistochemistry followed by confocal imaging. FINDINGS: We visualised perinuclear RNAscope signal for negative-sense SARS-CoV-2 RNA species in ciliated cells of the bronchiolar epithelium of a patient who died with COVID-19 in the hyperacute phase of the infection, and in ciliated cells of a primary culture of human airway epithelium that had been infected experimentally with SARS-CoV-2. In patients who died between 5 and 13 days after diagnosis of the infection, we detected RNAscope signal for positive-sense but not for negative-sense SARS-CoV-2 RNA species in pneumocytes, macrophages, and among debris in the alveoli. SARS-CoV-2 RNA levels decreased after a disease course of 2-3 weeks, concomitant with a histopathological change from exudative to fibroproliferative diffuse alveolar damage. Taken together, our confocal images illustrate the complexities stemming from traditional approaches in the literature to characterise cell tropism and visualise ongoing viral replication solely by the surrogate parameters of nucleocapsid-immunoreactive signal or in situ hybridisation for positive-sense SARS-CoV-2 RNA species. INTERPRETATION: Confocal imaging of human lung sections stained fluorescently with commercially available RNAscope probes for negative-sense SARS-CoV-2 RNA species enables the visualisation of viral replication at single-cell resolution during the acute phase of the infection in COVID-19. This methodology will be valuable for research on future SARS-CoV-2 variants and other respiratory viruses. FUNDING: Max Planck Society, Coronafonds UZ/KU Leuven, European Society for Organ Transplantation.

Effects of repeated infections with non-typeable Haemophilus influenzae on lung in vitamin D deficient and smoking mice.

BACKGROUND: In chronic obstructive pulmonary disease (COPD), exacerbations cause acute inflammatory flare-ups and increase the risk for hospitalization and mortality. Exacerbations are common in all disease stages and are often caused by bacterial infections e.g., non-typeable Heamophilus influenzae (NTHi). Accumulating evidence also associates vitamin D deficiency with the severity of COPD and exacerbation frequency. However, it is still unclear whether vitamin D deficiency when combined with cigarette smoking would worsen and prolong exacerbations caused by repeated infections with the same bacterial strain. METHODS: Vitamin D sufficient (VDS) and deficient (VDD) mice were exposed to nose-only cigarette smoke (CS) for 14 weeks and oropharyngeally instilled with NTHi at week 6, 10 and 14. Three days after the last instillation, mice were assessed for lung function, tissue remodeling, inflammation and immunity. The impact of VDD and CS on inflammatory cells and immunoglobulin (Ig) production was also assessed in non-infected animals while serum Ig production against NTHi and dsDNA was measured in COPD patients before and 1 year after supplementation with Vitamin D3. RESULTS: VDD enhanced NTHi eradication, independently of CS and complete eradication was reflected by decreased anti-NTHi Ig's within the lung. In addition, VDD led to an increase in total lung capacity (TLC), lung compliance (Cchord), MMP12/TIMP1 ratio with a rise in serum Ig titers and anti-dsDNA Ig's. Interestingly, in non-infected animals, VDD exacerbated the CS-induced anti-NTHi Ig's, anti-dsDNA Ig's and inflammatory cells within the lung. In COPD patients, serum Ig production was not affected by vitamin D status but anti-NTHi IgG increased after vitamin D3 supplementation in patients who were Vitamin D insufficient before treatment. CONCLUSION: During repeated infections, VDD facilitated NTHi eradication and resolution of local lung inflammation through production of anti-NTHi Ig, independently of CS whilst it also promoted autoantibodies. In COPD patients, vitamin D supplementation could be protective against NTHi infections in vitamin D insufficient patients. Future research is needed to decipher the determinants of dual effects of VDD on adaptive immunity. TRAIL REGISTRATION: ClinicalTrials, NCT00666367. Registered 23 April 2008, https://www.clinicaltrials.gov/ct2/show/study/NCT00666367 .

Oxygenated machine perfusion at room temperature as an alternative for static cold storage in porcine donor hearts.

BACKGROUND: There is a continued interest in ex situ heart perfusion as an alternative strategy for donor heart preservation. We hypothesize that oxygenated machine perfusion of donor hearts at a temperature that avoids both normothermia and deep hypothermia offers adequate and safe preservation. METHODS: Cardioplegia-arrested porcine donor hearts were randomly assigned to six hours of preservation using cold storage (CS, n = 5) or machine perfusion using an oxygenated acellular perfusate at 21°C (MP, n = 5). Subsequently, all grafts were evaluated using the Langendorff method for 120 min. Metabolic parameters and histology were analyzed. Systolic function was assessed by contractility and elastance. Diastolic function was assessed by lusitropy and stiffness. RESULTS: For both groups, in vivo baseline and post-Langendorff biopsies were comparable, as were lactate difference and myocardial oxygen consumption. Injury markers gradually increased and were comparable. Significant weight gain was seen in MP (p = 0.008). Diastolic function was not impaired in MP, and lusitropy was superior from 30 min up to 90 min of reperfusion. Contractility was superior in MP during the first hour of evaluation. CONCLUSION: We conclude that the initial functional outcome of MP-preserved hearts was transiently superior compared to CS, with no histological injury post-Langendorff. Our machine perfusion strategy could offer feasible and safe storage of hearts prior to transplantation. Future studies are warranted for further optimization.

Defining and predicting progression in non-IPF interstitial lung disease.

Randomized placebo-controlled trials demonstrated the efficacy of antifibrotic treatment in non-IPF progressive fibrosing ILD (fILD). Currently, there is no consensus on how progression should be defined and clinical data of non-IPF fILD patients in a real-world setting are scarce. Non-IPF fILD patients presenting at the University Hospitals Leuven between 2012 and 2016 were included. Different definitions of progression according to the selection criteria of the INBUILD, RELIEF and the uILD study were retrospectively evaluated at every hospital visit. Univariate and multivariate analyses were performed to identify predictors of progression and mortality. The study cohort comprised 120 patients; 68.3%, 54.2% and 65.8% had progressive disease based on the INBUILD, RELIEF and uILD study, respectively. A large overlap of progressive fILD patients according to the different clinical trials was observed. Median transplant-free survival time of progressive fILD patients was 3.9, 3.9, 3.8 years and the median time-to-progression after diagnosis was 2.0, 3.1 and 2.3 years according to the INBUILD, RELIEF and uILD study, respectively. We identified several predictors of mortality, but only an underlying diagnosis of HP and uILD was independently associated with progression. Our data show a high prevalence of progressive fibrosis among non-IPF fILD patients and a discrepancy between predictors of mortality and progression. Mortality rate in fILD is high and the identification of progressive disease is only made late during the disease course. Moreover, future treatment decisions will be based upon disease behavior. Therefore, more predictors of progressive disease are needed to guide treatment decisions in the future.

Early oseltamivir reduces risk for influenza-associated aspergillosis in a double-hit murine model.

Invasive pulmonary aspergillosis (IPA) is a life-threatening fungal infection occurring mainly in immunocompromised patients. We recently identified IPA as an emerging co-infection with high mortality in critically ill, but otherwise immunocompetent influenza patients. The neuraminidase inhibitor oseltamivir is the current standard-of-care treatment in hospitalized influenza patients; however, its efficacy in influenza-associated pulmonary aspergillosis (IAPA) is not known. Therefore, we have established an imaging-supported double-hit mouse model to investigate the therapeutic effect of oseltamivir on the development of IAPA. Immunocompetent mice received intranasal instillation influenza A or PBS followed by orotracheal inoculation with Aspergillus fumigatus 4 days later. Oseltamivir treatment or placebo was started at day 0, day 2, or day 4. Daily monitoring included micro-computed tomography and bioluminescence imaging of pneumonia and fungal burden. Non-invasive biomarkers were complemented with imaging, molecular, immunological, and pathological analysis. Influenza virus-infected immunocompetent mice developed proven airway IPA upon co-infection with Aspergillus fumigatus, whereas non-influenza-infected mice fully cleared Aspergillus, confirming influenza as a risk factor for developing IPA. Longitudinal micro-CT showed pulmonary lesions after influenza infection worsening after Aspergillus co-infection, congruent with bioluminescence imaging and histology confirming Aspergillus pneumonia. Early oseltamivir treatment prevented severe influenza pneumonia and mitigated the development of IPA and associated mortality. A time-dependent treatment effect was consistently observed with imaging, molecular, and pathological analyses. Hence, our findings underscore the importance of initiating oseltamivir as soon as possible, to suppress influenza infection and mitigate the risk of potentially lethal IAPA disease.

Connective Tissue Growth Factor Is Overexpressed in Explant Lung Tissue and Broncho-Alveolar Lavage in Transplant-Related Pulmonary Fibrosis.

Background: Connective tissue growth factor (CTGF) is an important mediator in several fibrotic diseases, including lung fibrosis. We investigated CTGF-expression in chronic lung allograft dysfunction (CLAD) and pulmonary graft-versus-host disease (GVHD). Materials and Methods: CTGF expression was assessed by quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry in end-stage CLAD explant lung tissue (bronchiolitis obliterans syndrome (BOS), n=20; restrictive allograft syndrome (RAS), n=20), pulmonary GHVD (n=9). Unused donor lungs served as control group (n=20). Next, 60 matched lung transplant recipients (BOS, n=20; RAS, n=20; stable lung transplant recipients, n=20) were included for analysis of CTGF protein levels in plasma and broncho-alveolar lavage (BAL) fluid at 3 months post-transplant, 1 year post-transplant, at CLAD diagnosis or 2 years post-transplant in stable patients. Results: qPCR revealed an overall significant difference in the relative content of CTGF mRNA in BOS, RAS and pulmonary GVHD vs. controls (p=0.014). Immunohistochemistry showed a significant higher percentage and intensity of CTGF-positive respiratory epithelial cells in BOS, RAS and pulmonary GVHD patients vs. controls (p<0.0001). BAL CTGF protein levels were significantly higher at 3 months post-transplant in future RAS vs. stable or BOS (p=0.028). At CLAD diagnosis, BAL protein content was significantly increased in RAS patients vs. stable (p=0.0007) and BOS patients (p=0.042). CTGF plasma values were similar in BOS, RAS, and stable patients (p=0.74). Conclusions: Lung CTGF-expression is increased in end-stage CLAD and pulmonary GVHD; and higher CTGF-levels are present in BAL of RAS patients at CLAD diagnosis. Our results suggest a potential role for CTGF in CLAD, especially RAS, and pulmonary GVHD.

Lung Functioning and Inflammation in a Mouse Model of Systemic Juvenile Idiopathic Arthritis.

Systemic juvenile idiopathic arthritis (sJIA) is an immune disorder characterized by fever, skin rash, arthritis and splenomegaly. Recently, increasing number of sJIA patients were reported having lung disease. Here, we explored lung abnormalities in a mouse model for sJIA relying on injection of IFN-γ deficient (IFN-γ KO) mice with complete Freund's adjuvant (CFA). Monitoring of lung changes during development of sJIA using microcomputer tomography revealed a moderate enlargement of lungs, a decrease in aerated and increase in non-aerated lung density. When lung function and airway reactivity to methacholine was assessed, gender differences were seen. While male mice showed an increased tissue hysteresivity, female animals were characterized by an increased airway hyperactivity, mirroring ongoing inflammation. Histologically, lungs of sJIA-like mice showed subpleural and parenchymal cellular infiltrates and formation of small granulomas. Flow cytometric analysis identified immature and mature neutrophils, and activated macrophages as major cell infiltrates. Lung inflammation in sJIA-like mice was accompanied by augmented expression of IL-1ß and IL-6, two target cytokines in the treatment of sJIA. The increased expression of granulocyte colony stimulating factor, a potent inducer of granulopoiesis, in lungs of mice was striking considering the observed neutrophilia in patients. We conclude that development of sJIA in a mouse model is associated with lung inflammation which is distinct to the lung manifestations seen in sJIA patients. Our observations however underscore the importance of monitoring lung disease during systemic inflammation and the model provides a tool to explore the underlying mechanism of lung pathology in an autoinflammatory disease context.

Involvement of Innate Lymphoid Cells and Dendritic Cells in a Mouse Model of Chemical-induced Asthma.

PURPOSE: Exposure to low concentrations of toluene diisocyanate (TDI) leads to immune-mediated chemical-induced asthma. The role of the adaptive immune system has already been thoroughly investigated; nevertheless, the involvement of innate immune cells in the pathophysiology of chemical-induced asthma is still unresolved. The aim of the study is to investigate the role of innate lymphoid cells (ILCs) and dendritic cells (DCs) in a mouse model for chemical-induced asthma. METHODS: On days 1 and 8, BALB/c mice were dermally treated (20 µL/ear) with 0.5% TDI or the vehicle acetone olive oil (AOO; 2:3). On days 15, 17, 19, 22 and 24, the mice received an oropharyngeal challenge with 0.01% TDI or AOO (1:4). One day after the last challenge, airway hyperreactivity (AHR) to methacholine was assessed, followed by an evaluation of pulmonary inflammation and immune-related parameters, including the cytokine pattern in bronchoalveolar lavage fluid, lymphocyte subpopulations of the lymph nodes and their ex vivo cytokine production profile, blood immunoglobulins and DC and ILC subpopulations in the lungs. RESULTS: Both DC and ILC2 were recruited to the lungs after multiple airway exposures to TDI, regardless of the prior dermal sensitization. However, prior dermal sensitization with TDI alone results in AHR and predominant eosinophilic airway inflammation, accompanied by a typical type 2 helper T (Th2) cytokine profile. CONCLUSIONS: TDI-induced asthma is mediated by a predominant type 2 immune response, with the involvement of adaptive Th2 cells. However, from our study we suggest that the innate ILC2 cells are important additional players in the development of TDI-induced asthma.

Overcome Double Trouble: Baloxavir Marboxil Suppresses Influenza Thereby Mitigating Secondary Invasive Pulmonary Aspergillosis.

Influenza-associated pulmonary aspergillosis (IAPA) is a global recognized superinfection in critically ill influenza patients. Baloxavir marboxil, a cap-dependent endonuclease inhibitor, is a newly approved anti-influenza therapeutic. Although the benefits as a treatment for influenza are clear, its efficacy against an influenza-A. fumigatus co-infection has yet to be determined. We investigated the therapeutic effect of baloxavir marboxil in a murine model for IAPA. Immunocompetent mice received intranasal instillation of influenza A followed by orotracheal inoculation with Aspergillus fumigatus 4 days later. Administration of baloxavir marboxil or sham was started at day 0, day 2 or day 4. Mice were monitored daily for overall health status, lung pathology with micro-computed tomography (µCT) and fungal burden with bioluminescence imaging (BLI). In vivo imaging was supplemented with virological, mycological and biochemical endpoint investigations. We observed an improved body weight, survival and viral clearance in baloxavir marboxil treated mice. µCT showed less pulmonary lesions and bronchial dilation after influenza and after Aspergillus co-infection in a treatment-dependent pattern. Furthermore, baloxavir marboxil was associated with effective inhibition of fungal invasion. Hence, our results provide evidence that baloxavir marboxil mitigates severe influenza thereby decreasing the susceptibility to a lethal invasive Aspergillus superinfection.

Small airway loss in the physiologically ageing lung: a cross-sectional study in unused donor lungs.

BACKGROUND: Physiological lung ageing is associated with a gradual decline in dynamic lung volumes and a progressive increase in residual volume due to diminished elastic recoil of the lung, loss of alveolar tissue, and lower chest wall compliance. However, the effects of ageing on the small airways (ie, airways <2·0 mm in diameter) remain largely unknown. By using a combination of ex-vivo conventional CT (resolution 1 mm), whole lung micro-CT (resolution 150 µm), and micro-CT of extracted cores (resolution 10 µm), we aimed to provide a multiresolution assessment of the small airways in lung ageing in a large cohort of never smokers. METHODS: For this cross-sectional study, we included donor lungs collected from 32 deceased never-smoking donors (age range 16-83 years). Ex-vivo CT and whole lung high-resolution CT (micro-CT) were used to determine total airway numbers, stratified by airway diameter. Micro-CT was used to assess the number, length, and diameter of terminal bronchioles (ie, the last generation of conducting airways); mean linear intercept; and surface density in four lung tissue cores from each lung, extracted using a uniform sampling approach. Regression ß coefficients are calculated using linear regression and polynomial models. FINDINGS: Ex-vivo CT analysis showed an age-dependent decrease in the number of airways of diameter 2·0 mm to less than 2·5 mm (ß coefficient per decade -0·119, 95% CI -0·193 to -0·045; R2=0·29) and especially in airways smaller than 2·0 mm in diameter (-0·158, -0·233 to -0·084; R2=0·47), between 30 and 80 years of age, but not of the larger (≥2·5 mm) diameter airways (-0·00781, -0·04409 to 0·02848; R2=0·0007). In micro-CT analysis of small airways, the total number of terminal bronchioles per lung increased until the age of 30 years, after which an almost linear decline in the number of terminal bronchioles was observed (ß coefficient per decade -2035, 95% CI -2818 to -1252; R2=0·55), accompanied by a non-significant increase in alveolar airspace size (6·44, -0·57 to 13·45, R2=0·10). Moreover, this decrease in terminal bronchioles was associated with the age-related decline of pulmonary function predicted by healthy reference values. INTERPRETATION: Loss of terminal bronchioles is an important structural component of age-related decline in pulmonary function of healthy, non-smoking individuals. FUNDING: Research Foundation-Flanders, KU Leuven, Parker B Francis Foundation, UGent, Canadian Institutes for Health.

Free Airway C4d after Lung Transplantation - A Quantitative Analysis of Bronchoalveolar Lavage Fluid.

In recent years, the utility of vascular complement factor 4d (C4d) deposition as diagnostic tool for antibody mediated rejection (AMR) after lung transplantation, has become a controversial issue. We aimed to pinpoint the problematic nature of C4d as biomarker with a simple experiment. We quantified C4d in broncho-alveolar lavage (BAL) of lung transplant patients with diverse post-transplant complications in 3 different settings of clinically clear cases of: 1/ chronic lung allograft dysfunction (CLAD); 2/ acute complications acute rejection (AR), lymphocytic bronchiolitis (LB), antibody-mediated rejection (AMR) and respiratory infection (INF); 3/ patients with parallel C4d immunostaining and Anti-HLA. All groups were compared to BAL of stable patients. C4d was measured via standard ELISA. C4d was increased in CLAD, predominantly in RAS (p = 0.0026) but not in BOS (p = 0.89). C4d was increased in all acute events, AR (p = 0.0025), LB (p < 0.0001), AMR (p = 0.0034), infections (p < 0.0001). In patients with parallel C4d immunostaining and serum HLA antibodies, C4d was increased in C4d-/HLA- (p = 0.0011); C4d-/HLA+ (p = 0.013); HLA+/C4d + (p = 0.0081). A correlation of systemic C-reactive protein (CRP) with C4d was found in all patients (r = 0.49; p < 0.0001). We hypothesize that free C4d in BAL may only be representative of a general immune response in the transplanted lung.

Common occurrence of Belerina virus, a novel paramyxovirus found in Belgian hedgehogs.

Common or European hedgehogs can be found throughout Western Europe. They are known carriers of a variety of parasitic and bacterial pathogens, and have also been shown to carry several viruses, including morbilli-like paramyxoviruses, although the pathogenic and zoonotic potential of some of these viruses has yet to be determined. We report here the discovery of a novel paramyxovirus in Belgian hedgehogs, named Belerina virus. The virus was detected by nanopore sequencing of RNA isolated from hedgehog tissue. Out of 147 animals screened in this study, 57 tested positive for Belerina virus (39%), indicating a high prevalence of this virus in the Belgian hedgehog population. Based on its divergence from other known paramyxovirus species, Belerina virus is thought to represent a new species in the family Paramyxoviridae. Phylogenetic analysis groups Belerina virus together with the bat-borne Shaan virus within the genus Jeilongvirus, although expanding the tree with partial genomes shows Belerina virus forming a separate subclade within this genus, alongside a yet-unnamed paramyxovirus isolated from a greater tube-nosed bat. In summary, we discuss the complete genome sequence of Belerina virus, a putative new paramyxovirus species commonly found in Belgian hedgehogs.

Longitudinal micro-computed tomography-derived biomarkers quantify non-resolving lung fibrosis in a silicosis mouse model.

In spite of many compounds identified as antifibrotic in preclinical studies, pulmonary fibrosis remains a life-threatening condition for which highly effective treatment is still lacking. Towards improving the success-rate of bench-to-bedside translation, we investigated in vivo µCT-derived biomarkers to repeatedly quantify experimental silica-induced pulmonary fibrosis and assessed clinically relevant readouts up to several months after silicosis induction. Mice were oropharyngeally instilled with crystalline silica or saline and longitudinally monitored with respiratory-gated-high-resolution µCT to evaluate disease onset and progress using scan-derived biomarkers. At weeks 1, 5, 9 and 15, we assessed lung function, inflammation and fibrosis in subsets of mice in a cross-sectional manner. Silica-instillation increased the non-aerated lung volume, corresponding to onset and progression of inflammatory and fibrotic processes not resolving with time. Moreover, total lung volume progressively increased with silicosis. The volume of healthy, aerated lung first dropped then increased, corresponding to an acute inflammatory response followed by recovery into lower elevated aerated lung volume. Imaging results were confirmed by a significantly decreased Tiffeneau index, increased neutrophilic inflammation, increased IL-13, MCP-1, MIP-2 and TNF-α concentration in bronchoalveolar lavage fluid, increased collagen content and fibrotic nodules. µCT-derived biomarkers enable longitudinal evaluation of early onset inflammation and non-resolving pulmonary fibrosis as well as lung volumes in a sensitive and non-invasive manner. This approach and model of non-resolving lung fibrosis provides quantitative assessment of disease progression and stabilization over weeks and months, essential towards evaluation of fibrotic disease burden and antifibrotic therapy evaluation in preclinical studies.

From Mouse to Man and Back: Closing the Correlation Gap between Imaging and Histopathology for Lung Diseases.

Lung diseases such as fibrosis, asthma, cystic fibrosis, infection and cancer are life-threatening conditions that slowly deteriorate quality of life and for which our diagnostic power is high, but our knowledge on etiology and/or effective treatment options still contains important gaps. In the context of day-to-day practice, clinical and preclinical studies, clinicians and basic researchers team up and continuously strive to increase insights into lung disease progression, diagnostic and treatment options. To unravel disease processes and to test novel therapeutic approaches, investigators typically rely on end-stage procedures such as serum analysis, cyto-/chemokine profiles and selective tissue histology from animal models. These techniques are useful but provide only a snapshot of disease processes that are essentially dynamic in time and space. Technology allowing evaluation of live animals repeatedly is indispensable to gain a better insight into the dynamics of lung disease progression and treatment effects. Computed tomography (CT) is a clinical diagnostic imaging technique that can have enormous benefits in a research context too. Yet, the implementation of imaging techniques in laboratories lags behind. In this review we want to showcase the integrated approaches and novel developments in imaging, lung functional testing and pathological techniques that are used to assess, diagnose, quantify and treat lung disease and that may be employed in research on patients and animals. Imaging approaches result in often novel anatomical and functional biomarkers, resulting in many advantages, such as better insight in disease progression and a reduction in the numbers of animals necessary. We here showcase integrated assessment of lung disease with imaging and histopathological technologies, applied to the example of lung fibrosis. Better integration of clinical and preclinical imaging technologies with pathology will ultimately result in improved clinical translation of (therapy) study results.

Cobalt exposure via skin alters lung immune cells and enhances pulmonary responses to cobalt in mice.

Cobalt has been associated with allergic contact dermatitis and occupational asthma. However, the link between skin exposure and lung responses to cobalt is currently unknown. We investigated the effect of prior dermal sensitization to cobalt on pulmonary physiological and immunological responses after subsequent challenge with cobalt via the airways. BALB/c mice received epicutaneous applications (25 µL/ear) with 5% CoCl2*6H2O (Co) or the vehicle (Veh) dimethyl sulfoxide (DMSO) twice; they then received oropharyngeal challenges with 0.05% CoCl2*6H2O or saline five times, thereby obtaining four groups: Veh/Veh, Co/Veh, Veh/Co, and Co/Co. To detect early respiratory responses noninvasively, we performed sequential in vivo microcomputed tomography (µCT). One day after the last challenge, we assessed airway hyperreactivity (AHR) to methacholine, inflammation in bronchoalveolar lavage (BAL), innate lymphoid cells (ILCs) and dendritic cells (DCs) in the lungs, and serum IgE. Compared with the Veh/Veh group, the Co/Co group showed increased µCT-derived lung response, increased AHR to methacholine, mixed neutrophilic and eosinophilic inflammation, elevated monocyte chemoattractant protein-1 (MCP-1), and elevated keratinocyte chemoattractant (KC) in BAL. Flow cytometry in the Co/Co group demonstrated increased DC, type 1 and type 2 conventional DC (cDC1/cDC2), monocyte-derived DC, increased ILC group 2, and natural cytotoxicity receptor-ILC group 3. The Veh/Co group showed only increased AHR to methacholine and elevated MCP-1 in BAL, whereas the Co/Veh group showed increased cDC1 and ILC2 in lung. We conclude that dermal sensitization to cobalt may increase the susceptibility of the lungs to inhaling cobalt. Mechanistically, this enhanced susceptibility involves changes in pulmonary DCs and ILCs.

Quantitative analysis of airway obstruction in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare, cystic lung disease with progressive pulmonary function loss caused by progressively proliferating LAM cells. The degree of airway obstruction has not been well investigated within the pathogenesis of LAM.Using a combination of ex vivo computed tomography (CT), microCT and histology, the site and nature of airway obstruction in LAM explant lungs was compared with matched control lungs (n=5 each). The total number of airways per generation, total airway counts, terminal bronchioles number and surface density were compared in LAM versus control.Ex vivo CT analysis demonstrated a reduced number of airways from generation 7 on (p<0.0001) in LAM compared with control, whereas whole-lung microCT analysis confirmed the three- to four-fold reduction in the number of airways. Specimen microCT analysis further demonstrated a four-fold decrease in the number of terminal bronchioles (p=0.0079) and a decreased surface density (p=0.0079). Serial microCT and histology images directly showed the loss of functional airways by collapse of airways on the cysts and filling of the airway by exudate.LAM lungs show a three- to four-fold decrease in the number of (small) airways, caused by cystic destruction which is the likely culprit for the progressive loss of pulmonary function.

Small airways pathology in idiopathic pulmonary fibrosis: a retrospective cohort study.

BACKGROUND: The observation that patients with idiopathic pulmonary fibrosis (IPF) can have higher than normal expiratory flow rates at low lung volumes led to the conclusion that the airways are spared in IPF. This study aimed to re-examine the hypothesis that airways are spared in IPF using a multiresolution imaging protocol that combines multidetector CT (MDCT), with micro-CT and histology. METHODS: This was a retrospective cohort study comparing explanted lungs from patients with severe IPF treated by lung transplantation with a cohort of unused donor (control) lungs. The donor control lungs had no known lung disease, comorbidities, or structural lung injury, and were deemed appropriate for transplantation on review of the clinical files. The diagnosis of IPF in the lungs from patients was established by a multidisciplinary consensus committee according to existing guidelines, and was confirmed by video-assisted thoracic surgical biopsy or by pathological examination of the contralateral lung. The control and IPF groups were matched for age, sex, height, and bodyweight. Samples of lung tissue were compared using the multiresolution imaging approach: a cascade of clinical MDCT, micro-CT, and histological imaging. We did two experiments: in experiment 1, all the lungs were randomly sampled; in experiment 2, samples were selected from regions of minimal and established fibrosis. The patients and donors were recruited from the Katholieke Universiteit Leuven (Leuven, Belgium) and the University of Pennsylvania Hospital (Philadelphia, PA, USA). The study took place at the Katholieke Universiteit Leuven, and the University of British Columbia (Vancouver, BC, Canada). FINDINGS: Between Oct 5, 2009, and July 22, 2016, explanted lungs from patients with severe IPF (n=11), were compared with a cohort of unused donor (control) lungs (n=10), providing 240 samples of lung tissue for comparison using the multiresolution imaging approach. The MDCT specimen scans show that the number of visible airways located between the ninth generation (control 69 [SD 22] versus patients with IPF 105 [33], p=0·0023) and 14th generation (control 9 [6] versus patients with IPF 49 [28], p<0·0001) of airway branching are increased in patients with IPF, which we show by micro-CT is due to thickening of their walls and distortion of their lumens. The micro-CT analysis showed that compared with healthy (control) lung anatomy (mean 5·6 terminal bronchioles per mL [SD 1·6]), minimal fibrosis in IPF tissue was associated with a 57% loss of the terminal bronchioles (mean 2·4 terminal bronchioles per mL [SD 1·0]; p<0·0001), the appearance of fibroblastic foci, and infiltration of the tissue by inflammatory immune cells capable of forming lymphoid follicles. Established fibrosis in IPF tissue had a similar reduction (66%) in the number of terminal bronchioles (mean 1·9 terminal bronchioles per mL [SD 1·4]; p<0·0001) and was dominated by increased airspace size, Ashcroft fibrosis score, and volume fractions of tissue and collagen. INTERPRETATION: Small airways disease is a feature of IPF, with significant loss of terminal bronchioles occuring within regions of minimal fibrosis. On the basis of these findings, we postulate that the small airways could become a potential therapeutic target in IPF. FUNDING: Katholieke Universiteit Leuven, US National Institutes of Health, BC Lung Association, and Genentech.