Stained Lung Sections Research Articles

Establishment of a stem cell administration imaging method in bleomycin-induced pulmonary fibrosis mouse models

Pulmonary fibrosis is a progressive disease caused by interstitial inflammation. Treatments are extremely scarce; therapeutic drugs and transplantation therapies are not widely available due to cost and a lack of donors, respectively. Recently, there has been a high interest in regenerative medicine and exponential advancements in stem cell-based therapies have occurred. However, a sensitive imaging technique for investigating the in vivo dynamics of transplanted stem cells has not yet been established and the mechanisms of stem cell-based therapy remain largely unexplored. In this study, we administered mouse adipose tissue-derived mesenchymal stem cells (mASCs) labeled with quantum dots (QDs; 8.0 nM) to a mouse model of bleomycin-induced pulmonary fibrosis in an effort to clarify the relationship between in vivo dynamics and therapeutic efficacy. These QD-labeled mASCs were injected into the trachea of C57BL/6 mice seven days after bleomycin administration to induce fibrosis in the lungs. The therapeutic effects and efficacy were evaluated via in vivo/ex vivo imaging, CT imaging, and H&E staining of lung sections. The QD-labeled mASCs remained in the lungs longer and suppressed fibrosis. The 3D imaging results showed that the transplanted cells accumulated in the peripheral and fibrotic regions of the lungs. These results indicate that mASCs may prevent fibrosis. Thus, QD labeling could be a suitable and sensitive imaging technique for evaluating in vivo kinetics in correlation with the efficacy of cell therapy.

MMP-3 mediates copper oxide nanoparticle-induced pulmonary inflammation and fibrosis

BackgroundThe increasing production and usage of copper oxide nanoparticles (Nano-CuO) raise human health concerns. Previous studies have demonstrated that exposure to Nano-CuO could induce lung inflammation, injury, and fibrosis. However, the potential underlying mechanisms are still unclear. Here, we proposed that matrix metalloproteinase-3 (MMP-3) might play an important role in Nano-CuO-induced lung inflammation, injury, and fibrosis.ResultsExposure of mice to Nano-CuO caused acute lung inflammation and injury in a dose-dependent manner, which was reflected by increased total cell number, neutrophil count, macrophage count, lactate dehydrogenase (LDH) activity, and CXCL1/KC level in bronchoalveolar lavage fluid (BALF) obtained on day 3 post-exposure. The time-response study showed that Nano-CuO-induced acute lung inflammation and injury appeared as early as day 1 after exposure, peaked on day 3, and ameliorated over time. However, even on day 42 post-exposure, the LDH activity and macrophage count were still higher than those in the control group, suggesting that Nano-CuO caused chronic lung inflammation. The Nano-CuO-induced pulmonary inflammation was further confirmed by H&E staining of lung sections. Trichrome staining showed that Nano-CuO exposure caused pulmonary fibrosis from day 14 to day 42 post-exposure with an increasing tendency over time. Increased hydroxyproline content and expression levels of fibrosis-associated proteins in mouse lungs were also observed. In addition, Nano-CuO exposure induced MMP-3 overexpression and increased MMP-3 secretion in mouse lungs. Knocking down MMP-3 in mouse lungs significantly attenuated Nano-CuO-induced acute and chronic lung inflammation and fibrosis. Moreover, Nano-CuO exposure caused sustained production of cleaved osteopontin (OPN) in mouse lungs, which was also significantly decreased by knocking down MMP-3.ConclusionsOur results demonstrated that short-term Nano-CuO exposure caused acute lung inflammation and injury, while long-term exposure induced chronic pulmonary inflammation and fibrosis. Knocking down MMP-3 significantly ameliorated Nano-CuO-induced pulmonary inflammation, injury, and fibrosis, and also attenuated Nano-CuO-induced cleaved OPN level. Our study suggests that MMP-3 may play important roles in Nano-CuO-induced pulmonary inflammation and fibrosis via cleavage of OPN and may provide a further understanding of the mechanisms underlying Nano-CuO-induced pulmonary toxicity.Graphical

Repeated Silica exposures lead to Silicosis severity via PINK1/PARKIN mediated mitochondrial dysfunction in mice model

Background and objectivesSilicosis, one of the occupational health illnesses is caused by inhalation of crystalline silica. Deposition of extracellular matrix and fibroblast proliferation in lungs are linked to silicosis development. Mitochondrial dysfunction plays critical role in some diseases, but how these processes progress and regulated in silicosis, remains limited. Detailed study of silica induced pulmonary fibrosis in mouse model, its progression and severity may be helpful in designing future therapeutic strategies. MethodsIn present study, mice model of silicosis has been developed after repeated silica exposures which may closely resemble clinical symptoms of silicosis in human. In addition to efficiently mimicking the acute/chronic transformation processes of silicosis, this is practical and efficient in terms of time and output, which avoids mechanical injury to the upper respiratory tract due to surgical interventions. Sonicated sterile silica suspension (120 mg/kg) was administered through intranasal route thrice a week at regular intervals (21, 28 and 35 days). ResultsPresence of minute to larger silicotic nodules in H&E-stained lung sections were observed in all silica induced model groups. Enhanced ECM deposition was noted in MT stained lung sections of silica exposure groups as compared to control which were confirmed by significantly higher MMP9 expression levels and hydroxyproline content in silica 35 days group. Increase in Reactive oxygen species (ROS), inflammatory cell recruitment mainly, neutrophils and macrophage were observed in all three silica exposure groups. Transmission electron microscopic analysis has confirmed presence of many aberrant shaped mitochondria (swollen, round shape) in 35 days model where autophagosomes were minimum. Western blot analysis of mitophagy and autophagy markers such as Pink1, Parkin, Cytochrome c, SQSTM1/p62, the ratio of light chain LC3B II/LC3B I was found higher in 21 and 28 days which were significantly reduced in 35 days silica model. ConclusionsHigher MMP9 activity and MMP9 /TIMP1 ratio demonstrate excessive extracellular matrix damage and deposition in 35 days model. Significantly reduced expressions of autophagy and mitophagy markers have also confirmed progression in fibrosis severity and its association with repeated silica exposures in 35 days model group.

Abstract 3132: Examining The Role Of The Lung Endothelial Cell Anti-apoptotic Protein C-iap2 In Schistosomiasis-associated Pulmonary Hypertension

Introduction: Schistosomiasis-associated Pulmonary Hypertension (Sch-PH) is the most common form of group I PH worldwide and remains with no targeted therapy. Previously, we observed that the loss of the anti-inflammatory protein caveolin-1 (Cav-1) in lung endothelial cells (EC) contributed to the survival of the abnormal EC phenotype in Sch-PH. Abnormal cell survival has been associated with some members of the inhibitor of apoptosis protein (IAP) family, but their role in Sch-PH is unknown. Hypothesis: The major etiological agent of Sch-PH, i.e., S. mansoni , may contribute to the abnormal lung EC phenotype via altered IAP family expression, specifically the inducible member c-IAP2. Methods/Methods: To test this hypothesis, we first quantified constitutive c-IAP1 and inducible c-IAP2 expression on lung samples from male and female control and pre-clinical S. mansoni egg-exposed mice (240 eggs/gram body weight intraperitoneally followed by intravenous injection of 175 eggs/gram body weight). Then, the specific contribution of EC-c-IAP2 expression to the progression of the disease was evaluated in our new mouse model ( Cdh5.CreERT2;cIAP1 -/- ,cIAP2 fl/fl - Cdh5.CreERT2;cIAP1 +/- ,cIAP2 +/fl ). EC- Cav1 -/- mice were also evaluated to test the contribution of EC-Cav-1 to lung c-IAP2 expression. Results/Discussion: Western blot and immunohistochemistry analysis revealed a reduction in c-IAP2 expression in the whole lung tissue of S. mansoni egg-exposed mice compared to controls (1.21 ± 0.12; 0.88 ± 0.06, control and egg-exposed mice respectively; p<0.05; n=6). No difference was observed in c-IAP1 expression. Preliminary data indicate that genetic ablation of c-IAP2 in EC heterozygous mice promoted a mild increase in microvascular thickness and area in Masson’s Trichrome -stained lung sections. Consistent with our previous findings, c-IAP2 expression was also significantly impaired when Cav-1 expression was absent in lung ECs, suggesting that EC-Cav-1 may contribute to reduced lung c-IAP2 expression, leading to the severe vascular remodeling that underlies the development of Sch-PH. Conclusion: Our data is uncovering whether c-IAP2 expression is a novel and specific molecular target for future therapeutic approaches against chronic Sch-PH.

The pulmonary neuroepithelial body-vagal sensory nerve axis is altered in a mouse model of bronchopulmonary dysplasia (BPD)

Breathing control problems, like apnea of prematurity, are a common complication in preterm infants with bronchopulmonary dysplasia (BPD), but the underlying factors remain incompletely understood. A subpopulation of pulmonary vagal afferent nerve fibers (Calb1+) innervate CO2 and O2 sensitive clusters of lung neuroendocrine cells (neuroepithelial bodies (NEBs)) located along conducting airways. Calb1+ fibers provide direct sensory input to the brainstem that putatively limit tidal volume. NEBs are hyperplastic in BPD infants and may increase activity of inhibitory Calb1+ vagal fibers. Herein, we sought to determine if the NEB-Calb1 lung-brainstem axis is altered in association with breathing control in BPD. BPD in mice was established by exposure to chronic neonatal (postnatal day (P) 0-14) hyperoxia (Hx; 70% O2; n=20) or normoxia (Nx; n=17). Consistent with human BPD, Hx caused alveolar simplification supported by a reduction in alveolar density and alveoli (p<0.05; separate t-tests) in P22 H&E-stained lung sections (n=5-7/group). Like human infants at risk for BPD, Hx mice adapted to increased dead space (caused by reduction in alveoli) by hyperventilating (p<0.05; generalized linear modeling accounting for fat free mass and heat production, measured by NMR and respirometry; n=10-24/group). This was facilitated by an increase in tidal volume that was significantly reduced at P14 but increased at P17 and P21 (n=19-28) (Tukey’s post-hoc: p<0.05). NEBs and associated innervation (vagal and dorsal root ganglia) were next assessed in subsets of mice. First, IT instillation of WGA-594 lectin into subsets of mice (n=9 mice/18 ganglia per group) and subsequent quantification of 594+ neurons per vagal ganglia revealed fewer neurons (p=0.07; t-test) in Hx mice (110.8 ± 6.9 vs 95.2 ± 4.9). Second, CGRP+ innervation to NEBs is reduced in Hx mice (Quantification of CGRP+ nerve fibers only: 17740.4 +/- 4126 vs 10474.6 +/- 2140 +CGRP pixels; p=0.12 n=5-7/group). Third, there was greater frequency of NEBs per unit airway (p<0.05; 0.306 ± 0.03 vs 0.401 ± 0.03) with a tendency for larger NEBs (p=0.06; 9.14 ± 0.7 vs 11.93 ± 1.3) indicating NEB hyperplasia. Together, these data indicate individual NEB innervation is reduced in BPD but that total NEB innervation may remain unchanged. If this is true for Calb1+ vagal fibers remain untested. To functionally assess Calb1-vagal fibers we activated, with DCZ, the excitatory synthetic Gq-DREAD receptor (DTR) expressed in Calb1cre mice via microinjection of AAV-Cre-Gq-mCherry into the left vagal ganglia. DCZ but not vehicle caused a major increase in peak inspiratory flow during acute hypoxic challenges compared to vehicle treated mice (Pre-DMSO, Post-DMSO, Pre-DCZ = ~1ml/sec vs 4ml/sec post-DCZ; p<0.05, n=4/group). Collection of vagal ganglia confirms DTR expression but spillover of DTR is present. Repeating these studies with diluted AAV-Cre-Gq in control and BPD mice are ongoing. Together, our data verify that hyperoxia induced BPD in mice causes a similar reduction in alveoli and compensation to increased dead space by hyperventilation as observed in human BPD. Additionally, BPD structurally alters the NEB-vagal axis but whether these changes are functionally significant to nCOB is being addressed with ongoing studies. AHA 20CDA35310121, Advancing Healthier Wisconsin Endowment. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Estrogen Metabolites Modulate Airway Hyperreactivity in a Murine Model of Asthma

Objective: Airway hyperreactivity (AHR) and remodeling are the key contributors to airway changes in asthma resulting in airway narrowing and resistance to airflow. Sex-steroid signaling, especially estrogen’s differential role in asthma undoubtedly questions its prime involvement in regulating airway structure and function in context to asthma. Evidence from cardiovascular (CV) and reproductive systems suggests that estrogen metabolites particularly 2-hydroxyestradiol (2HE) and 16α-hydroxyestradiol (16αHE2) influence a multitude of cellular functions. Accordingly, the aim of this study is to determine how estrogen metabolites regulate AHR and remodeling using a mouse model of asthma. Methods: WT C57BL/6J (male and female) mice were procured from Jackson Laboratory. Experimental animals were administered with PBS, 2-HE (2 mg/kg), 16αHE2 (2 mg/kg) with/without MA intranasally on alternate days for 28 days. On day 29, mice were anesthetized and subjected to SCIREQ flexiVent to measure Lung mechanics like airway resistance (Rrs), elastance (Ers), and compliance (Crs). Following this, broncho-alveolar lavage (BAL) was performed for differential leukocyte count (DLC) and lung tissue samples processed for histology using hematoxylin and eosin (H&E) staining and Sirius-red and fast-green (SRFG). Results: MA-challenged group increased airway Rrs and reduced Crs in response to methacholine (MCh) in both male and female mice with profound effects in female mice. Animals exposed to MA and administered with 16αHE2 showed significant increase the Rrs while 2-HE downregulated the MA effect. The exacerbated effect of 16αHE2 on Rrs was significantly higher in MA-challenged female mice compared to male mice. Histology study including H&E and SRFG stained lung sections show increased ASM mass in 16αHE2 exposed mixed MA challenged mice. Whereas 2HE reversed MA induced effect. DLC in BAL showed increased neutrophils and eosinophils in MA challenged mice, this was attenuated by 2HE. Conclusion: This study highlights the distinct role of estrogen metabolites in regulating airway hyperresponsiveness and remodeling in the context of asthma. Thus, providing possible therapeutic potential for observed sex differences in asthma. Acknowledgment: Supported by NIH grant R01-HL146705 (Sathish). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

FGF10 attenuates allergic airway inflammation in asthma by inhibiting PI3K/AKT/NF-κB pathway

BackgroundThe effect of fibroblast growth factor 10 (Fgf10) against allergic asthma has remained unclear, despite its importance in lung development and homeostasis maintenance. The purpose of this study was to investigate the protective effect and potential mechanism of Fgf10 on asthma. MethodHouse Dust Mite (HDM)-induced asthma mice were administered recombinant Fgf10 intranasally during activation. Flow cytometry and ELISA were performed to determine type of inflammatory cells and type 2 cytokines levels in bronchoalveolar lavage fluid (BALF). Hematoxylin and eosin (H&E) and periodic acid - Schiff (PAS) staining of lung sections were conducted to evaluate histopathological assessment. Transcriptome profiling was analyzed using RNA-seq, followed by bioinformatics and network analyses to investigate the potential mechanisms of Fgf10 in asthma. RT-qPCR was also used to search for and validate differentially expressed genes in human Peripheral Blood Mononuclear Cells (PBMCs). ResultsExogenous administration of Fgf10 alleviated HDM-induced inflammation and mucus secretion in lung tissues of mice. Fgf10 also significantly inhibited the accumulation of eosinophils and type 2 cytokines (IL-4, IL-5, and IL-13) in BALF. The PI3K/AKT/NF-κB pathway may mediate the suppressive impact of Fgf10 on the asthma inflammation. Through RNA-seq analysis, the intersection of 71 differentially expressed genes (DEGs) was found between HDM challenge and Fgf10 treatment. GO and KEGG enrichment analyses indicated a strong correlation between the DEGs and different immune response. Immune infiltration analysis predicted the differential infiltration of five types of immune cells, such as NK cells, dendritic cells, monocytes and M1 macrophages. PPI analysis determined hub genes such as Irf7, Rsad2, Isg15 and Rtp4. Interestingly, above genes were consistently altered in human PBMCs in asthmatic patients. ConclusionAsthma airway inflammation could be attenuated by Fgf10 in this study, suggesting that it could be a potential therapeutic target.

Ethanol exacerbates pulmonary complications after burn injury in mice, regardless of frequency of ethanol exposures

Burn injuries are associated with significant morbidity and mortality, and lungs are the most common organ to fail. Interestingly, patients with alcohol intoxication at the time of burn have worse clinical outcomes, including pulmonary complications. Using a clinically relevant murine model, we have previously reported that episodic ethanol exposure before burn exacerbated lung inflammation. Specifically, intoxicated burned mice had worsened pulmonary responses, including increased leukocyte infiltration and heightened levels of CXCL1 and IL-6. Herein, we examined whether a single binge ethanol exposure before scald burn injury yields similar pulmonary responses. C57BL/6 male mice were given ethanol (1.2 g/kg) 30 min before a 15 % total body surface area burn. These mice were compared to a second cohort given episodic ethanol binge for a total of 6 days (3 days ethanol, 4 days rest, 3 days ethanol) prior to burn injury. 24 h after burn, histopathological examination of lungs were performed. In addition, survival, and levels of infiltrating leukocytes, CXCL1, and IL-6 were quantified. Episodic and single ethanol exposure before burn decreased survival compared to burn only mice and sham vehicle mice, respectively (p < 0.05). However, no difference in survival was observed between burned mice with single and episodic ethanol binge. Examination of H&E-stained lung sections revealed that regardless of ethanol binge frequency, intoxication prior to burn worsened pulmonary inflammation, evidenced by elevated granulocyte accumulation and congestion, relative to burned mice without any ethanol exposure. Levels of infiltrating granulocyte in the lungs were significantly higher in burned mice with both episodic and single ethanol intoxication, compared to burn injury only (p < 0.05). In addition, there was no difference in the granulocyte count between single and ethanol binge mice with burn injury. Neutrophil chemoattractant CXCL1 levels in the lung were similarly increased following single and episodic ethanol exposure prior to burn compared to burn alone (22-fold and 26-fold respectively, p < 0.05). Lastly, we assessed pulmonary IL-6, which revealed that irrespective of frequency, ethanol exposure combined with burn injury raised pro-inflammatory cytokine IL-6 in the lungs relative to burn mice. Again, we did not find any difference in the amount of IL-6 in lungs of burned mice with single and episodic ethanol intoxication. Taken altogether, these data demonstrate that both single and episodic exposure to ethanol prior to burn injury similarly worsens pulmonary inflammation. These results suggest that ethanol-induced exacerbation of the pulmonary responses to burn injury is due to presence of ethanol at the time of injury rather than longer-term effects of ethanol exposure.

The NEB-P2ry1 lung-brainstem axis and the control of breathing in a mouse model of bronchopulmonary dysplasia

Breathing control problems, like apnea of prematurity, are a common complication in preterm infants with bronchopulmonary dysplasia (BPD), but the underlying factors remain incompletely understood. A subpopulation of pulmonary vagal afferent nerve fibers (P2ry1+) innervate CO2 and O2 sensitive clusters of lung neuroendocrine cells (neuroepithelial bodies (NEBs)) located along conducting airways. P2ry1 fibers provide direct sensory input to the brainstem and inhibit breathing upon stimulation. NEBs are hyperplastic in BPD infants and may increase activity of inhibitory P2ry1 vagal fibers. Herein, we sought to determine if the NEB-P2ry1 lung-brainstem axis is altered in association with breathing control in BPD. BPD in mice was induced by exposure to chronic neonatal (postnatal day (P) 0-14) hyperoxia (Hx; 70% O2; n=20) or normoxia (Nx; n=17). Consistent with human BPD, Hx caused alveolar simplification supported by a reduction in alveolar density (p&lt;0.05) and alveoli (p&lt;0.05) in P22 H&amp;E-stained lung sections. Hx mice spent more time apneic at P14 and P17 (p&lt;0.05) in room air and acute hypoxia. Overall (main effect), room air minute ventilation (VE) was reduced in HX mice (p&lt;0.05). Further, VE was reduced during acute hypoxia at P14 and P17 (not P21) with an overall reduction of breathing in Hx mice (p&lt;0.05). From a subset of mice (n=4-5/group) VCO2, VO2, and heat production at rest were greater in P21 Hx mice during room air, suggesting the Hx mice were hypoventilating. Immunofluorescent staining of CGRP (hallmark NEB marker), Ki67 (proliferation), and SEZ6L2 (new NEB cell-membrane marker) in Hx and Nx lungs were imaged and analyzed by a blinded investigator. Consistent with clinical findings, Hx mice had hyperplastic NEBs (8.6 ± 0.45 vs 10.7 ± 1.3 cells/NEB; p&lt;0.05) while only 2 of 143 NEBs expressed Ki67, suggesting no proliferation at P22. CGRP co-localized with SEZ6L2 in both Nx and Hx lungs. A subset of mice received intratracheal delivery of 1ug SEZ6L2-Saporin toxin to lesion NEBs and assess their function. 4 days after SEZ-Saporin treatment breathing frequency was elevated by 11% ± 5% in room air and by 7% ± 5% during acute hypoxia but not in PBS-treated, adult mice (n=3/group) suggesting NEBs have a mild inhibitory influence under normal conditions. Lastly, wireless optogenetic stimulation of P2ry1 vagal fibers in unanesthetized adult control mice (n=3) increased room air breathing by 20% ± 1% and drive to breathe by 36% ± 19% compared to pre-stim breathing. Together, our data demonstrate that hyperoxia-induced BPD alters the control of breathing which may in part be mediated via a unique lung-brainstem axis comprised of hyperplastic NEBs causing greater inhibition of excitatory P2ry1 vagal fibers. The NEB-P2ry1 axis may serve as a novel target for improving breathing in BPD. GCM: Advancing Healthier Wisconsin REAC Award, AHA 20CDA35310121; MRH: HL122358; JLG: DK133121, HL134850, and HL084207; JLS: DK133121 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Modeling the Effects of Cypermethrin Toxicity on Ovalbumin-Induced Allergic Pneumonitis Rats: Macrophage Phenotype Differentiation and p38/STAT6 Signaling Are Candidate Targets of Pirfenidone Treatment

Although the classic form of asthma is characterized by chronic pneumonitis with eosinophil infiltration and steroid responsivity, asthma has multifactorial pathogenesis and various clinical phenotypes. Previous studies strongly suggested that chemical exposure could influence the severity and course of asthma and reduce its steroid responsiveness. Cypermethrin (CYP), a common pesticide used in agriculture, was investigated for the possible aggravation of the ovalbumin (OVA)-induced allergic pneumonitis and the possible induction of steroid resistance in rats. Additionally, it was investigated whether pirfenidone (PFD) could substitute dexamethasone, as an alternative treatment option, for the induced steroid resistance. Fifty-six male Wistar albino rats were randomly divided into seven groups: control, PFD alone, allergic pneumonitis, CYP alone, allergic pneumonitis/CYP-exposed, allergic pneumonitis/CYP/dexamethasone (Dex), and allergic pneumonitis/CYP/PFD-treated groups. Allergic pneumonitis was induced by three intraperitoneal OVA injections administered once a week, followed by an intranasal OVA instillation challenge. CYP (25 mg/kg/d), Dex (1 mg/kg/d), and PFD (100 mg/kg/d) were administered orally from day 15 to the end of the experiment. Bronchoalveolar lavage fluid (BALF) was analyzed for cytokine levels. Hematoxylin and eosin (H&E) and periodic acid Schiff (PAS)-stained lung sections were prepared. Immunohistochemical identification of p38 MAPK and lung macrophages was performed. The inflammatory/oxidative status of the lung and PCR-quantification of the STAT6, p38 MAPK, MUC5AC, and IL-13 genes were carried out. The allergic pneumonitis-only group showed eosinophil-mediated inflammation (p < 0.05). Further CYP exposure aggravated lung inflammation and showed steroid-resistant changes, p38 activation, neutrophil-mediated, M1 macrophage-related inflammation (p < 0.05). All changes were reversed (p < 0.05) by PFD, meanwhile not by dexamethasone treatment. Pirfenidone could replace dexamethasone treatment in the current rat model of CYP-induced severe steroid-resistant asthma via inhibiting the M1 macrophage differentiation through modulation of the STAT6/p38 MAPK pathway.

Back cover story: Eur. J. Immunol. 11'22

European Journal of ImmunologyVolume 52, Issue 11 Back CoverFree Access Back cover story: Eur. J. Immunol. 11'22 First published: 03 November 2022 https://doi.org/10.1002/eji.202270116AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Our back cover features an H&E staining of lung sections from 8-month-old mice with a thymic epithelial cell (TEC)-specific Trappc1 deficiency. Apparent lymphocyte infiltration indicated that Trappc1 deletion intrinsically caused defective TEC development and maturation, which led to abnormal T cell development and autoimmune disorder in mice. The image is taken from Dong et al., where the authors positioned Trappc1 as a key intrinsic determinant molecule of TEC development and maturation, and provided strong evidence for the critical role of interorganelle traffic and ER homeostasis in TEC development. Volume52, Issue11November 2022 RelatedInformation

Psychological Stress of aged mice has long-term impact on Mycobacterium tuberculosis infection by altering composition of lung T cell populations

Abstract Aging is a major risk factor for chronic infections, including tuberculosis (TB). Elderly TB patients also suffer from elevated levels of psychological stress. It is not known how psychological stress impacts TB pathogenesis. To study the effects of stress on Mycobacterium tuberculosis (M.tb) we used a social disruption stress (SDR) model which involves repeated social defeat in subordinate mice. Young (3 month) and Old (18 month) C57BL/6 mice were subjected to SDR and infected with M.tb. Mice were sacrificed at 30 and 60 days post infection and M.tb burden analyzed by CFU assay and lung pathology by HE staining of lung sections. At day 30, M.tb burden was lower in control old mice compared to young mice and SDR did not significantly alter M.tb burden. However, at day 60, SDR in old mice significantly increased M.tb burden compared to SDR in young mice, indicating that SDR in old mice has a detrimental effect on the ability to control the infection. Our gene expression analysis revealed that T cell genes (JAK3 and LCP2) in the lungs of old SDR infected mice were increased but not in young SDR mice, suggesting that SDR in old mice increased T cell recruitment in the lung. Additionally, we found that Tbet (Th1) and GATA3 (Th2) gene expression were unchanged by SDR, in contrast the expression of RORγT (Th17) was increased by SDR in old mice at day 60. Also, mRNA levels of IL-10 at day 60 were increased by SDR in old mice, but not in young mice, while SDR decreased IFN-γ in both young and old mice. To further define T cell populations, we are analyzing lung sections by confocal microscopy. In conclusion, our studies suggest that psychological stress prior to M.tb prior to infection has long-term impact by altering composition of T cell populations in the lung. Supported by NIH grants (PO1-AG051428, AI146252 and AI146690)

Chronic Exposure to Electronic Cigarettes Induces Lung Oxidative Stress, Inflammation, Fibrosis, and Impaired DNA Repair

BackgroundElectronic cigarettes (e‐cig) have been marketed as a safer alternative to tobacco cigarette smoking (TCS). While there are far less toxic compounds in e‐cig vape (ECV) than in tobacco cigarette smoke, detectable levels of toxicants are still present. Multiple in vitro and short‐term in vivo studies on e‐cig‐induced lung cell damage have been reported; however, questions remain regarding the health effects of chronic e‐cig use on the lungs. Therefore, studies were performed in a chronic mouse exposure model to investigate the deleterious effects of long‐term ECV exposure on the lungs.MethodsC57/BL6 male mice were exposed for 2 hours/day, 5 days/week to either air (control group; 25 mice) or ECV generated from e‐cig liquid containing 24 mg/ml nicotine (e‐cig group; 25 mice) for 16 weeks. At the end of the exposure, animals were euthanized, lungs were lavaged, then the recovered bronchoalveolar lavage fluid (BALF) was prepared to assess the percentages of inflammatory cells in BALF. Fixed lung sections were also prepared and stained with hematoxylin and eosin (H &amp; E) or Masson’s trichrome stain to assess the degree of lung inflammation and fibrosis; respectively. Superoxide radical generation was measured in frozen lung sections using DHE. Western blotting (WB) and immunofluorescence (IF) in lung tissue were used to detect the levels of nitrotyrosine, and the expression of the DNA repair enzymes XPC and OGG1.ResultsThe number of inflammatory cells present in the BALF, including neutrophils, macrophages, and eosinophils, was significantly higher in ECV‐exposed mice compared to that with air exposure. In the ECV‐exposed mice, H &amp; E stained lung sections showed clear inflammatory infiltrates with lymphoid aggregates of lymphocytes, plasma cells and macrophages that stretch from the perivascular and peribronchiolar areas into adjacent septal adipose tissue. Masson’s trichrome stained slides showed increase in connective tissue around airways and bronchioles in e‐cig exposed mice, indicating the presence of pulmonary fibrosis. Using the fluorescence‐based superoxide probe DHE, significantly increased superoxide generation was observed in the lungs of ECV exposed mice, compared to control. This DHE‐derived fluorescence was largely quenched by preincubation with the SOD mimetic MnTBAP, confirming that the observed fluorescence was largely derived from superoxide. WB and IF showed elevated levels of nitrotyrosine. Reduced expression of the DNA damage repair proteins XPC and OGG1 were seen in lung tissue of the ECV exposed mice compared to the controls.ConclusionChronic ECV exposure induced lung inflammation, pulmonary fibrosis, and increased superoxide generation. ECV exposure increased oxidative stress and inflammation in the lungs while decreasing the levels of DNA damage repair proteins. These findings suggest that chronic e‐cig use can trigger oxidative stress with inflammatory lung damage and impaired DNA repair that together may predispose to neoplastic transformation and cancer onset.

IL23R on myeloid cells is involved in murine pulmonary granuloma formation

Purpose of the Study The involvement of the IL-23/IL23R pathway is well known in the disease pathogenesis of sarcoidosis and other inflammatory diseases. To date, the pathogenic mechanism of IL-23 is most notably described on CD4+ Th17 lymphocytes. However, the function of the IL23R on myeloid cells in sarcoidosis is poorly understood. Thus, the aim of the study is to investigate the role of the IL23R on myeloid cell in pulmonary granuloma formation. Methods: We generated IL23RLysMCre mice lacking the IL23R gene in myeloid cells. The importance of IL23R in myeloid cells for the development of sarcoidosis was studied in a mouse model of inflammatory lung granuloma formation through embolization of PPD from Mycobacterium bovis-coated Sepharose beads into previously PPD-immunized mice. In addition the function of IL23R on myeloid cells was studied in LPS or IFNγ stimulated BMDMs and BMDCs. The mRNA and protein expression levels of relevant cytokines were analyzed by RT-PCR (TaqMan) and ELISA. The composition of immune cells in BALF was quantified by flow cytometry and alteration in granuloma sizes were observed by H&E stained lung sections. Results: Mycobacterium Ag-elicted pulmonary granulomas tend to be smaller in IL23RLysMCre mice and NF-κB dependent Th1 cytokines in the murine lungs are reduced compared to wildtype mice. In line, we observed that IL23R-deficient bone marrow-derived macrophages show a reduced production of Th1 cytokines after LPS stimulation. Conclusion: We here for the first time demonstrate a role for IL23R on myeloid cells in pulmonary inflammation and granuloma formation. Our findings provide essential insights in the pathogenesis of inflammatory lung diseases like sarcoidosis, which might be useful for the development of novel therapeutics targeting distinct immunological pathways like IL-23/IL23R.

Calycosin Alleviates Injury in Airway Epithelial Cells Caused by PM 2.5 Exposure via Activation of AMPK Signalling.

Methods Phospho-AMP-activated protein kinase (p-AMPK) and AMP-activated protein kinase (AMPK) were detected by western blot. Immunofluorescence staining was used to validate changes in the levels of nuclear factor kappa B (NF-кB) p65 nuclear translocation. Mice were administered intraperitoneally with calycosin one hour before anaesthesia and endotracheal instillation of PM 2.5. The extent of lung injury was evaluated in the H&E-stained lung sections. Apoptotic cells were detected by TUNEL staining. Results Administration of calycosin was increased in PM 2.5-treated B2B cells in a dose-dependent manner in vitro. Fluorescence signals from anti-NF-кB p65 were increased in nuclei of cells pretreated with calycosin. The level of p-AMPK was increased by calycosin in vitro and in vivo. After pretreatment with compound C, the inhibitory effects of calycosin on cytotoxicity, levels of inflammatory cytokines and p-AMPK, and levels of NF-кB p65 nuclear translocation were not significantly decreased in vitro or in vivo. Conclusions Calycosin effectively decreased the release of inflammatory cytokines and alleviated injury caused by PM 2.5. These effects were mediated through activation of AMPK to suppress NF-κB signalling.

Tim-3 is dispensable for allergic inflammation and respiratory tolerance in experimental asthma.

T cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3) has been described as a transmembrane protein, expressed on the surface of various T cells as well as different cells of innate immunity. It has since been associated with Th1 mediated autoimmune diseases and transplantation tolerance studies, thereby indicating a possible role of this receptor in counter-regulation of Th2 immune responses. In the present study we therefore directly examined the role of Tim-3 in allergic inflammation and respiratory tolerance. First, Tim-3-/- mice and wild type controls were immunized and challenged with the model allergen ovalbumin (OVA) to induce an asthma-like phenotype. Analysis of cell numbers and distribution in the bronchoalveolar lavage (BAL) fluid as well as lung histology in H&E stained lung sections demonstrated a comparable degree of eosinophilic inflammation in both mouse strains. Th2 cytokine production in restimulated cell culture supernatants and serum IgE and IgG levels were equally increased in both genotypes. In addition, cell proliferation and the distribution of different T cell subsets were comparable. Moreover, analysis of both mouse strains in our respiratory tolerance model, where mucosal application of the model allergen before immunization, prevents the development of an asthma-like phenotype, revealed no differences in any of the parameters mentioned above. The current study demonstrates that Tim-3 is dispensable not only for the development of allergic inflammation but also for induction of respiratory tolerance in mice in an OVA-based model.

Abstract PO-082: Automated tumor segmentation, grading, and analysis of tumor heterogeneity in preclinical models of lung adenocarcinoma

Abstract Preclinical mouse models of lung adenocarcinoma are invaluable for the discovery of molecular drivers of tumor formation, progression, and therapeutic resistance. Histological analyses of these preclinical models require significant investments of time and training to ensure accuracy and consistency. Analysis by a clinical pathologist is the gold standard in this approach, but may be difficult to obtain due to the cost and availability of their services. As an alternative we have developed a digital pathology tool to identify, segment, grade, and analyze tumors in mouse models of lung adenocarcinoma. This convolutional neural network (CNN) model, based on ResNet18, was trained to classify normal lung tissue, normal airways, and the different grades (1 – 4) of lung adenocarcinoma from 100,000 224 × 224 pixel image patches (~16,000 patches per class). Our training dataset was constructed from whole slide images of hematoxylin and eosin stained lung sections from 4 different mouse models of lung adenocarcinoma with oncogenic Kras (KrasG12D/+), in combination with oncogenic p53 mutations (KrasG12D/+; p53R172H/+ and KrasG12D/+;p53R270H/+), or with the loss of the tumor suppressive TAp73 (KrasG12D/+;TAp7fltd/fltd). Our CNN demonstrated a strong correspondence with human pathologists on our holdout dataset, achieving a micro-F1 score of 0.81 on a pixel-by-pixel basis. As a test of our CNN, we analyzed two mouse models to better understand the role of TAp73 in lung adenocarcinoma: KrasG12D/+ (“K”) and KrasG12D/+;TAp73fltd/fltd (“TK”). Both human raters and our CNN reported a significant increase in the tumor burden of the compound mutant “TK” mice compared to the single mutant “K” mice. According to our CNN, this increased tumor burden was driven primarily by an increase in tumor size and not an increased number of tumors in “TK” mice. Because our CNN can assign different grades to regions within the same image patch and tumor, we also uncovered a high degree of intratumor heterogeneity that was not reported by the human pathologists, who are trained to assign one grade to a single tumor with a bias for the highest grade present in a given tumor. The finer grading resolution allowed our CNN to uncover the increased tumor size observed in the “TK” mice was due to expansion of Grade 2 regions (characterized by enlarged nuclei without irregular shape) within tumors that would be considered a higher grade by pathologists. Our CNN also provides a detailed map of tumor grades overlaid on the H&amp;E images used for analysis, allowing for precise targeting of regions within tumors with other assays. We are currently utilizing these outputs in conjunction with other assays, such as spatial transcriptomic analysis and immunohistochemistry, to investigate the molecular mechanisms that underlie the expansion of Grade 2 tumor regions in “TK” mice. Future work will expand this tool into a multidimensional digital pathology pipeline that can accelerate current investigations and reveal new therapeutic targets and prognostic markers. Citation Format: John H. Lockhart, Hayley D. Ackerman, Kyubum Lee, Mahmoud Abdalah, Andrew Davis, Nicole Montey, Theresa Boyle, James Saller, Ayensur Keske, Kay Hänggi, Brian Ruffell, Olya Stringfield, Aik Choon Tan, Elsa R. Flores. Automated tumor segmentation, grading, and analysis of tumor heterogeneity in preclinical models of lung adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Artificial Intelligence, Diagnosis, and Imaging; 2021 Jan 13-14. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(5_Suppl):Abstract nr PO-082.

Live Imaging of the Lung.

Live imaging is critical to determining the dynamics and spatial interactions of cells within the tissue environment. In the lung, this has proven to be difficult due to the motion brought about by ventilation and cardiac contractions. A previous version of this Current Protocols in Cytometry article reported protocols for imaging ex vivo live lung slices and the intact mouse lung. Here, we update those protocols by adding new methodologies, new approaches for quantitative image analysis, and new areas of potential application. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Live imaging of lung slices Support Protocol 1: Staining lung sections with fluorescent antibodies Basic Protocol 2: Live imaging in the mouse lung Support Protocol 2: Intratracheal instillations Support Protocol 3: Intravascular instillations Support Protocol 4: Monitoring vital signs of the mouse during live lung imaging Support Protocol 5: Antibodies Support Protocol 6: Fluorescent reporter mice Basic Protocol 3: Quantification of neutrophil-platelet aggregation in pulmonary vasculature Basic Protocol 4: Quantification of platelet-dependent pulmonary thrombosis Basic Protocol 5: Quantification of pulmonary vascular permeability.

Abstract 13869: Inflammatory Changes in the Lungs of Rats Exposed to E-cigarettes Without Marijuana, Vitamin E Oil, or Nicotine

Background: Recently, the clinical entity of EVALI (E-cigarette or vaping product use associated lung injury) was described and has similarities to COVID -19 pneumonia. We determined in a quantitative manner the inflammatory changes in the lungs associated with EVALI in a rodent model that does not include use of marijuana, vitamin E oil, or nicotine, which had been implicated as a cause. Methods: Adult Sprague Dawley rats were exposed to either pure air (n=5) or electronic cigarette (eC) vapor (propylene glycol/vegetable glycerin, tobacco flavoring) using a device with a nickel-chromium alloy (NC) heating element at 70 Watts (n=6) for 2 hours. The e-liquid did not contain nicotine, marijuana, or Vitamin E oil. Rats were euthanized within a few days of exposure and H&amp;E-stained lung sections were assessed. Inflammatory cells were counted from 10 random areas per section at 20х magnification. Results: Alveolar structure appeared normal in the air group (Figure, panel C); the eC group using the NC heating element showed alveolar inflammation (D). The numbers of inflammatory cells per high power field in the lung parenchyma were significantly greater in the rats exposed to eC using NC heating element (67.5 ± 44.5) compared to the air group (6.7 ± 0.3; P &lt; 0.001, Kruskal-Wallis test). The predominant cell type was mononuclear and secondary were neutrophils. Other features included accumulation of fibrin and inflammatory cells in the lumen of the trachea (A); thickened alveolar walls; and red blood cell congestion (D). Inflammation of the nasal passages was also observed (B). Exposure of rats to e cigarettes using a stainless steel heating element did not result in EVALI, and the amount of inflammatory cells was closer to air exposed rats (7.2± 0.4). Conclusion: Vaping using a nichrome heating element at high power leads to respiratory tract pathology and a significant increase in the number of inflammatory cells in the alveoli. EVALI occurred without marijuana, vitamin E oil or nicotine.

Abstract 15303: Dimethylarginine Dimethylaminohydrolase-1 is Not Involved in Chronic Hypoxic Pulmonary Hypertension and Right Ventricular Hypertrophy in Mice

Introduction: Chronic hypoxia causes persistent pulmonary vasoconstriction and leads to pulmonary hypertension and right ventricular hypertrophy. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthesis; its level increases in hypoxia concomitantly with reduced activity of dimethylarginine dimethylaminohydrolases (DDAH-1 and DDAH-2), the enzymes metabolizing ADMA. DDAH knockout models may therefore help to understand the pathophysiological roles of this enzyme and its substrate, ADMA, in the development of hypoxia-associated pulmonary hypertension. Hypothesis: We hypothesized that DDAH1 knock-out mice have an attenuated hypoxia-induced elevation of ADMA and reduced right ventricular hypertrophy. Methods: DDAH1 knock-out mice (KO) and their wild-type littermates (WT) were subjected to normoxia (NX) or hypoxia (HX) during 21 days. We measured ADMA concentration in plasma, DDAH1 and DDAH2 expression in the lung, right ventricular hypertrophy by the Fulton index, cardiomyocyte hypertrophy by dystrophin staining of heart tissues, and muscularization of pulmonary arterioles by CD31 and α-actin staining of lung sections. Results: DDAH1 KO mice had higher ADMA concentration than WT under NX (2.31±0.33 μmol/l vs. 1.20±0.17 μmol/l; p &lt; 0.05). ADMA significantly increased in WT-HX (to 1.74±0.86 μmol/l; p &lt; 0.05 vs. normoxia), whilst it did not further increase in KO-HX (2.58±0.58 μmol/l; p = n.s.). This was paralleled by a 38±13% reduction in DDAH1 mRNA but not DDAH2 mRNA expression, and reduced DDAH protein expression. We observed right ventricular hypertrophy under hypoxia in both, WT and KO mice, with no significant differences between both genotypes. Further, cardiomyocyte hypertrophy and pulmonary arteriolar muscularization were significantly increased by hypoxia, but not significantly different between WT and KO mice. Conclusions: We conclude that chronic hypoxia causes an elevation of ADMA, which impairs NO production and leads to endothelial dysfunction and vasoconstriction. Downregulation of DDAH expression and activity may be involved in this; however, knockout of DDAH1 does not modify the pathophysiological changes in remodeling of the pulmonary vasculature and the right ventricle.