Mouse Lung Research Articles

Apigenin is an anoikis sensitizer with strong anti-metastatic properties in experimental breast cancer

Loss of susceptibility to anoikis signals is a crucial step in metastasis. Anoikis resistance therefore represents a promising adjuvant therapeutic target for cancer management. In this study, we have conducted a rationalized screening to search for novel leading anoikis sensitizer from daily foods. Among 19 tested dietary phytochemicals, the best results were obtained with apigenin, a natural component of celery. Phenotypically, apigenin sensitized breast cancer cells to anoikis, lowered the number of circulating tumor cells, and protected against breast cancer metastasis to lung in mice. Mechanistically, we demonstrated that the thromboxane A2 (TXA2)-TXA2 receptor (TP) axis has a critical role in acquired anoikis resistance by activating PI3K-Akt signaling pathway. Blockage of TXA2 signaling up-regulated p53 as well as its target gene p21, caused a G1 phase arrest, and finally led to apoptosis in breast cancer cells. TXA2 level was positively correlated with breast cancer cell anoikis rate, and apigenin significantly inhibited TXA2 biosynthesis in vitro and in vivo. Collectively, we identified apigenin as a potent anoikis sensitizer with anti-metastatic properties in a mouse model of breast cancer, and these findings might provide a rationale for introducing apigenin supplementation to breast cancer patients.

Spiramycin-chitosan Nanoparticles Decline Parasite Burden and Renovate Patent Histopathological Changes in Liver and Lung in Mice Experimentally Infected with Acute Toxoplasmosis

Spiramycin-chitosan Nanoparticles Decline Parasite Burden and Renovate Patent Histopathological Changes in Liver and Lung in Mice Experimentally Infected with Acute Toxoplasmosis

Helminth infection induces a distinct subset of CD101hi lung tissue-infiltrating eosinophils that are differentially regulated by type 2 cytokines.

Eosinophils play divergent roles in health and disease, contributing to both immunoregulatory and proinflammatory responses. Helminth infection is strongly associated with eosinophilia and the induction of the type 2 cytokines interleukin (IL)-5, IL-4 and IL-13. This study aimed to elucidate the heterogeneity of pulmonary eosinophils in response to helminth infection and the roles of IL-5, IL-4 and IL-13 in driving pulmonary eosinophil responses. Using the murine helminth model Nippostrongylus brasiliensis (Nb), we characterize a subtype of eosinophils, defined by high expression of CD101, that is induced in the lungs of Nb-infected mice and are phenotypically distinct from lung eosinophils that express low levels of CD101. Strikingly, we show that the two eosinophil subtypes have distinct anatomical localization within the lung: CD101low eosinophils are predominantly localized in the lung vasculature, whereas Nb-induced CD101hi eosinophils are predominantly localized in the extravascular lung niche. We show that CD101hi eosinophils are also induced across other models of pulmonary infection and inflammation, including a nonlung-migrating helminth infection, house dust mite-induced allergic inflammation and influenza infection. Furthermore, we demonstrate that the induction of CD101hi tissue eosinophils is independent of IL-5 and IL-4 signaling, but is dependent on intact IL-13 signaling. These results suggest that IL-13 produced during helminth infection and other disease states promotes a pulmonary tissue-infiltrating program in eosinophils defined by high expression of CD101.

Macrophage biomimetic nanoparticle-targeted functional extracellular vesicle micro-RNAs revealed via multiomics analysis alleviate sepsis-induced acute lung injury

Patients who suffer from sepsis typically experience acute lung injury (ALI). Extracellular vesicles (EVs) contain miRNAs, which are potentially involved in ALI. However, strategies to screen more effective EV-miRNAs as therapeutic targets are yet to be elucidated. In this study, functional EV-miRNAs were identified based on multiomics analysis of single-cell RNA sequencing of targeted organs and serum EV (sEV) miRNA profiles in patients with sepsis. The proportions of neutrophils and macrophages were increased significantly in the lungs of mice receiving sEVs from patients with sepsis compared with healthy controls. Macrophages released more EVs than neutrophils. MiR-125a-5p delivery by sEVs to lung macrophages inhibited Tnfaip3, while miR-221-3p delivery to lung neutrophils inhibited Fos. Macrophage membrane nanoparticles (MM NPs) loaded with an miR-125a-5p inhibitor or miR-221-3p mimic attenuated the response to lipopolysaccharide (LPS)-induced ALI. Transcriptome profiling revealed that EVs derived from LPS-stimulated bone marrow-derived macrophages (BMDMs) induced oxidative stress in neutrophils. Blocking toll-like receptor, CXCR2, or TNFα signaling in neutrophils attenuated the oxidative stress induced by LPS-stimulated BMDM-EVs. This study presents a novel method to screen functional EV-miRNAs and highlights the pivotal role of macrophage-derived EVs in ALI. MM NPs, as delivery systems of key sEV-miRNA mimics or inhibitors, alleviated cellular responses observed in sepsis-induced ALI. This strategy can be used to reduce septic organ damage, particularly lung damage, by targeting EVs.

CircADAMTS6 via stabilizing CAMK2A is involved in smoking-induced emphysema through driving M2 macrophage polarization

Cigarette smoke (CS), an indoor environmental pollutant, is a prominent risk factor for emphysema, which is a pathological feature of chronic obstructive pulmonary disease (COPD). Emerging function of circRNAs in immune responses and disease progression shed new light to explore the pathogenesis of emphysema. In this research, we demonstrated, by single-cell RNA sequencing (scRNAseq), that the ratio of M2 macrophages were increased in lung tissues of humans and mice with smoking-related emphysema. Further, our data showed that circADAMTS6 was associated with cigarette smoke extract (CSE)-induced M2 macrophage polarization. Mechanistically, in macrophages, circADAMTS6 stabilized CAMK2A mRNA via forming a circADAMTS6/IGF2BP2/CAMK2A RNA-protein ternary complex to activate CREB, which drives M2 macrophage polarization and leads to emphysema. In addition, in macrophages of mouse lung tissues, downregulation of circADAMTS6 reversed M2 macrophage polarization, the proteinase/anti-proteinase imbalance, and the elastin degradation, which protecting against CS-induced emphysema. Moreover, for macrophages and in a model with co-cultured lung organoids, the target of circADAMTS6 restored the growth of lung organoids compared to CSE-treated macrophages. Our results also demonstrated that, for smokers and COPD smokers, elevation of circADAMTS6 negatively correlated with lung function. Overall, this study reveals a novel mechanism for circADAMTS6-driven M2 macrophage polarization in smoking-related emphysema and postulates that circADAMTS6 could serve as a diagnostic and therapeutic marker for smoking-related emphysema.

Immune Responses and Protection Profiles in Mice Induced by Subunit Vaccine Candidates Based on the Extracellular Domain Antigen of Respiratory Syncytial Virus G Protein Combined with Different Adjuvants.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease of infants and older people. There is an urgent need for safe and effective vaccines against RSV infection. In this study, we analyzed the effects of the immune response and protection with the RSV recombinant G protein extracellular domain (Gecto) combined with various adjuvants as novel subunit vaccines in mice. All groups receiving RSV Gecto combined with adjuvants exhibited robust humoral and cellular immunity compared to those receiving an adjuvant alone or inactivated RSV vaccine. The greatest effect was observed in mice receiving Gecto combined with a CpG ODN + Alum salt adjuvant, resulting in the highest production of neutralizing antibodies against both RSV A and B subtypes, G-specific IgG and IFN-γ production in splenocytes, and interleukin-2 and interferon-γ expression in CD4+ T cells. Significant humoral and cellular immune responses were observed in mice immunized with Gecto combined with AddaS03™ or cyclosporin A adjuvants. The vaccine containing the AddaS03™ adjuvant showed significantly high expression of interleukin-4 in CD4+ T cells. Cross-protection against a challenge with either RSV A or B subtypes was observed in the Gecto plus adjuvant groups, resulting in a significant decrease in viral load and reduced pathological damage in the mouse lungs. These findings offer valuable insights into the development and application of recombinant RSV G-subunit vaccines with adjuvants.

Leveraging Integrated RNA Sequencing to Decipher Adrenomedullin's Protective Mechanisms in Experimental Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease commonly affecting premature infants, with limited therapeutic options and increased long-term consequences. Adrenomedullin (Adm), a proangiogenic peptide hormone, has been found to protect rodents against experimental BPD. This study aims to elucidate the molecular and cellular mechanisms through which Adm influences BPD pathogenesis using a lipopolysaccharide (LPS)-induced model of experimental BPD in mice. Bulk RNA sequencing of Adm-sufficient (wild-type or Adm+/+) and Adm-haplodeficient (Adm+/-) mice lungs, integrated with single-cell RNA sequencing data, revealed distinct gene expression patterns and cell type alterations associated with Adm deficiency and LPS exposure. Notably, computational integration with cell atlas data revealed that Adm-haplodeficient mouse lungs exhibited gene expression signatures characteristic of increased inflammation, natural killer (NK) cell frequency, and decreased endothelial cell and type II pneumocyte frequency. Furthermore, in silico human BPD patient data analysis supported our cell type frequency finding, highlighting elevated NK cells in BPD infants. These results underscore the protective role of Adm in experimental BPD and emphasize that it is a potential therapeutic target for BPD infants with an inflammatory phenotype.

Glyoxal-methyl-ethylene sulfonic acid fixative enhances the fixation of cytoskeletal structures for Förster resonance energy transfer measurements.

Förster resonance energy transfer (FRET) serves as a tool for measuring protein-protein interactions using various sensor molecules. The tension sensor module relies on FRET technology. In our study, this module was inserted within the actinin molecule to measure the surface tension of the cells. Given that the decay curve of FRET efficiency correlates with surface tension increase, precise and accurate efficiency measurement becomes crucial. Among the methods of FRET measurements, FRET efficiency remains the most accurate if sample fixation is successful. However, when cells were fixed with 4% paraformaldehyde (PFA), the actinin-FRET sensor diffused across the cytoplasm; this prompted us to explore fixation method enhancements. Glyoxal fixative has been reported to improve cytoskeletal morphologies compared to PFA. However, it was not known whether glyoxal fits FRET measurements. Glyoxal necessitates an acetic acid solution for fixation; however, acidic conditions could compromise fluorescence stability. We observed that the pH working range of glyoxal fixative aligns closely with MES (methyl-ethylene sulfonic acid) Good's buffer. Initially, we switched the acidic solution for MES buffer and optimized the fixation procedure for in vitro and in vivo FRET imaging. By comparing FRET measurements on hydrogels with known stiffness to tumor nodules in mouse lung, we estimated in vivo stiffness. The estimated stiffness of cancerous tissue was harder than the reported stiffness of smooth muscle. This discovery shed lights on how cancer cells perceive environmental stiffness during metastasis.

Imaging the Granzyme Mediated Host Immune Response to Viral and Bacterial Pathogens In Vivo Using Positron Emission Tomography.

Understanding how the host immune system engages complex pathogens is essential to developing therapeutic strategies to overcome their virulence. While granzymes are well understood to trigger apoptosis in infected host cells or bacteria, less is known about how the immune system mobilizes individual granzyme species in vivo to combat diverse pathogens. Toward the goal of studying individual granzyme function directly in vivo, we previously developed a new class of radiopharmaceuticals termed "restricted interaction peptides (RIPs)" that detect biochemically active endoproteases using positron emission tomography (PET). In this study, we showed that secreted granzyme B proteolysis in response to diverse viral and bacterial pathogens could be imaged with [64Cu]Cu-GRIP B, a RIP that specifically targets granzyme B. Wild-type or germline granzyme B knockout mice were instilled intranasally with the A/PR/8/34 H1N1 influenza A strain to generate pneumonia, and granzyme B production within the lungs was measured using [64Cu]Cu-GRIP B PET/CT. Murine myositis models of acute bacterial (E. coli, P. aeruginosa, K. pneumoniae, and L. monocytogenes) infection were also developed and imaged using [64Cu]Cu-GRIP B. In all cases, the mice were studied in vivo using mPET/CT and ex vivo via tissue-harvesting, gamma counting, and immunohistochemistry. [64Cu]Cu-GRIP B uptake was significantly higher in the lungs of wild-type mice that received A/PR/8/34 H1N1 influenza A strain compared to mice that received sham or granzyme B knockout mice that received either treatment. In wild-type mice, [64Cu]Cu-GRIP B uptake was significantly higher in the infected triceps muscle versus normal muscle and the contralateral triceps inoculated with heat killed bacteria. In granzyme B knockout mice, [64Cu]Cu-GRIP B uptake above the background was not observed in the infected triceps muscle. Interestingly, live L. monocytogenes did not induce detectable granzyme B on PET, despite prior in vitro data, suggesting a role for granzyme B in suppressing their pathogenicity. In summary, these data show that the granzyme response elicited by diverse human pathogens can be imaged using PET. These results and data generated via additional RIPs specific for other granzyme proteases will allow for a deeper mechanistic study analysis of their complex in vivo biology.

Substance P Promotes Leukocyte Infiltration in the Liver and Lungs of Mice with Sepsis: A Key Role for Adhesion Molecules on Vascular Endothelial Cells.

Substance P (SP), encoded by the Tac1 gene, has been shown to promote leukocyte infiltration and organ impairment in mice with sepsis. Neurokinin-1 receptor (NK1R) is the major receptor that mediates the detrimental impact of SP on sepsis. This investigation studied whether SP affects the expression of adhesion molecules, including intercellular cell adhesion molecule-1 (ICAM1) and vascular cell adhesion molecule-1 (VCAM1) on vascular endothelial cells in the liver and lungs, contributing to leukocyte infiltration in these tissues of mice with sepsis. Sepsis was induced by caecal ligation and puncture (CLP) surgery in mice. The actions of SP were inhibited by deleting the Tac1 gene, blocking NK1R, or combining these two methods. The activity of myeloperoxidase and the concentrations of ICAM1 and VCAM1 in the liver and lungs, as well as the expression of ICAM1 and VCAM1 on vascular endothelial cells in these tissues, were measured. The activity of myeloperoxidase and the concentration of ICAM1 and VCAM1 in the liver and lungs, as well as the expression of ICAM1 and VCAM1 on vascular endothelial cells in these tissues, increased in mice with CLP surgery-induced sepsis. Suppressing the biosynthesis of SP and its interactions with NK1R attenuated CLP surgery-induced alterations in the liver and lungs of mice. Our findings indicate that SP upregulates the expression of ICAM1 and VCAM1 on vascular endothelial cells in the liver and lungs, thereby increasing leukocyte infiltration in these tissues of mice with CLP surgery-induced sepsis by activating NK1R.

Impairment of Gal-9 and Tim-3 crosstalk between Tregs and Th17 cells drives tobacco smoke-induced airway inflammation.

Overexpression of T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) on T cells has been observed in smokers. However, whether and how galectin-9 (Gal-9)/TIM-3 signal between T-regulatory cells (Tregs) and type 17 helper (Th17) cells contributes to tobacco smoke-induced airway inflammation remains unclear. Here, we aimed to explore the role of the Gal-9/TIM-3 signal between Tregs and Th17 cells during chronic tobacco smoke exposure. Tregs phenotype and the expression of TIM-3 on CD4+ T cells were detected in a mouse model of experimental emphysema. The role of TIM-3 in CD4+ T cells was explored in a HAVCR2-/- mouse model and in mice that received recombinant anti-TIM3. The crosstalk between Gal-9 and Tim-3 was evaluated by coculture Tregs with effector CD4+ T cells. We also invested the expression of Gal-9 in Tregs in patients with COPD. Our study revealed that chronic tobacco smoke exposure significantly reduces the frequency of Tregs in the lungs of mice and remarkably shapes the heterogeneity of Tregs by downregulating the expression of Gal-9. We observed a pro-inflammatory but restrained phenotypic transition of CD4+ T cells after tobacco smoke exposure, which was maintained by TIM-3. The restrained phenotype of CD4+ T cells was perturbed when TIM-3 was deleted or neutralised. Tregs from the lungs of mice with emphysema displayed a blunt ability to inhibit the differentiation and proliferation of Th17 cells. The inhibitory function of Tregs was partially restored by using recombinant Gal-9. The interaction between Gal-9 and TIM-3 inhibits the differentiation of Th17 cells and promotes apoptosis of CD4+ T cells, possibly by interfering with the expression of retinoic acid receptor-related orphan receptor gamma t. The expression of Gal-9 in Tregs was reduced in patients with COPD, which was associated with Th17 response and lung function. These findings present a new paradigm that impairment of Gal-9/Tim-3 crosstalk between Tregs and Th17 cells during chronic tobacco smoke exposure promotes tobacco smoke-induced airway/lung inflammation.

Protective effects of growth hormone – Releasing hormone antagonists in the lungs of septic mice

Growth hormone-releasing hormone antagonists (GHRHAnt) have been associated with antitumor and antioxidative activities. The present study investigates for the first time the effects of those compounds towards pro-inflammatory cytokine expression in a murine model of cecal ligation and puncture (CLP) – induced sepsis. The results indicate that GHRHAnt JV-1-36 significantly suppressed IL-1α, IL-6, and pSTAT3 activation in septic lungs. Moreover, GHRHAnt treatment reduced bronchoalveolar lavage fluid (BALF) protein concentration, suggesting a protective effect of that compound in sepsis-induced lung edema. Based on those findings, it is suggested that GHRHAnt may represent an exciting new therapeutic possibility in sepsis-induced endotoxemia and lung injury.

Skeletal muscle BMAL1 is necessary for transcriptional adaptation of local and peripheral tissues in response to endurance exercise training

ObjectiveIn this investigation, we addressed the contribution of the core circadian clock factor, BMAL1, in skeletal muscle to both acute transcriptional responses to exercise and transcriptional remodeling in response to exercise training. Additionally, we adopted a systems biology approach to investigate how loss of skeletal muscle BMAL1 altered peripheral tissue homeostasis as well as exercise training adaptations in iWAT, liver, heart, and lung of male mice. MethodsCombining inducible skeletal muscle specific BMAL1 knockout mice, physiological testing and standardized exercise protocols, we performed a multi-omic analysis (transcriptomics, chromatin accessibility and metabolomics) to explore loss of muscle BMAL1 on muscle and peripheral tissue responses to exercise. ResultsMuscle-specific BMAL1 knockout mice demonstrated a blunted transcriptional response to acute exercise, characterized by the lack of upregulation of well-established exercise responsive transcription factors including Nr4a3 and Ppargc1a. Six weeks of exercise training in muscle-specific BMAL1 knockout mice induced significantly greater and divergent transcriptomic and metabolomic changes in muscle. Surprisingly, liver, lung, inguinal white adipose and heart showed divergent exercise training transcriptomes with less than 5% of ‘exercise-training’ responsive genes shared for each tissue between genotypes. ConclusionsOur investigation has uncovered the critical role that BMAL1 plays in skeletal muscle as a key regulator of gene expression programs for both acute exercise and training adaptations. In addition, our work has uncovered the significant impact that altered exercise response in muscle and its likely impact on the system plays in the peripheral tissue adaptations to exercise training. Our work also demonstrates that if the muscle adaptations diverge to a more maladaptive state this is linked to increased gene expression signatures of inflammation across many tissues. Understanding the molecular targets and pathways contributing to health vs. maladaptive exercise adaptations will be critical for the next stage of therapeutic design for exercise mimetics.

Targeting lysyl oxidase like 2 attenuates OVA-induced airway remodeling partly via the AKT signaling pathway

BackgroundAirway epithelium is an important component of airway structure and the initiator of airway remodeling in asthma. The changes of extracellular matrix (ECM), such as collagen deposition and structural disturbance, are typical pathological features of airway remodeling. Thus, identifying key mediators that derived from airway epithelium and capable of modulating ECM may provide valuable insights for targeted therapy of asthma.MethodsThe datasets from Gene Expression Omnibus database were analyzed to screen differentially expressed genes in airway epithelium of asthma. We collected bronchoscopic biopsies and serum samples from asthmatic and healthy subjects to assess lysyl oxidase like 2 (LOXL2) expression. RNA sequencing and various experiments were performed to determine the influences of LOXL2 knockdown in ovalbumin (OVA)-induced mouse models. The roles and mechanisms of LOXL2 in bronchial epithelial cells were explored using LOXL2 small interfering RNA, overexpression plasmid and AKT inhibitor.ResultsBoth bioinformatics analysis and further experiments revealed that LOXL2 is highly expressed in airway epithelium of asthmatics. In vivo, LOXL2 knockdown significantly inhibited OVA-induced ECM deposition and epithelial-mesenchymal transition (EMT) in mice. In vitro, the transfection experiments on 16HBE cells demonstrated that LOXL2 overexpression increases the expression of N-cadherin and fibronectin and reduces the expression of E-cadherin. Conversely, after silencing LOXL2, the expression of E-cadherin is up-regulated. In addition, the remodeling and EMT process that induced by transforming growth factor-β1 could be enhanced and weakened after LOXL2 overexpression and silencing in 16HBE cells. Combining the RNA sequencing of mouse lung tissues and experiments in vitro, LOXL2 was involved in the regulation of AKT signaling pathway. Moreover, the treatment with AKT inhibitor in vitro partially alleviated the consequences associated with LOXL2 overexpression.ConclusionsTaken together, the results demonstrated that epithelial LOXL2 plays a role in asthmatic airway remodeling partly via the AKT signaling pathway and highlighted the potential of LOXL2 as a therapeutic target for airway remodeling in asthma.

Preclinical analysis of the efficacy of near-infrared photoimmunotherapy and anti-PD-1 antibody combination therapy for treating CD73-positive lung cancer.

e14521 Background: The CD73/adenosine pathway, which converts extracellular ATP to adenosine, is emerging as a promising target for immunotherapy in various cancers. In the tumor microenvironment, hypoxia triggers upregulation of adenosine-activating molecules, including CD73, a membrane-anchored enzyme expressed on cancer cells. CD73 induces the expression of PD-1 and CTLA4, and all of this leads to immunosuppression. Therefore, we focused utilizing near-infrared photoimmunotherapy (NIR-PIT) to kill CD73-expressing cells and alleviate immunosuppression. NIR-PIT is an innovative cancer treatment approach which involves irradiating NIR light after the administrated antibody-photosensitizer conjugate binds to the target cells. Methods: In vitro, anti-CD73 antibody-IR700 conjugate was prepared and added to the CMT167 and LLC cell lines, which were irradiated with NIR light under a microscope. In vivo, a mouse model was established using the CMT167 cell line. The mice were divided into 6 groups: 1) NIR-PIT group against CD73; 2) anti-PD-1 antibody group; 3) combined treatment group of 1) and 2); 4) control group; 5) anti-CD73 antibody-IR700 conjugate group; and 6) near-infrared light irradiation group. Tumor volume and survival rate were compared among these groups. Additionally, after 2 weeks of treatment, tumors in the four groups 1) to 4) above were pathologically evaluated using two different AI-equipped software programs. Results: Microscope observation revealed that NIR-PIT induced immunogenic cell death of CD73-positive mouse lung cancer cell lines in vitro. In vivo, the effect of NIR-PIT on this mouse model was also demonstrated. Although previous studies have shown the efficacy of treatment with anti-CD73 antibodies, the present study compared the CD73-IR700 conjugate group to the NIR-PIT to CD73 group, and the latter group showed better therapeutic outcomes. Additionally, the combined treatment group displayed the highest anti-tumor efficacy in terms of survival and tumor volume. Pathological evaluation of the tumor interior showed a significant increase in tumor-infiltrating lymphocytes compared to the other groups. Additionally, the percentage of tumor cells was significantly reduced, while the percentage of stroma, including fibrous tissue, was significantly increased. In line with other studies that have already reported the additive effect of combination therapy with anti-CD73 antibodies and immune checkpoint inhibitors, the present study showed that the combination therapy resulted in further activation of immune cells and enhanced the therapeutic effect. Conclusions: CD73 represents a promising target for NIR-PIT, with potential for enhanced effectiveness when used in combination with immune checkpoint inhibitors.

Effect of Toll-like Receptor-3 Antagonist on Viral Asthma Exacerbations Via a TLR3/dsRNA Complex Pathway

Background: The Toll-like receptor-3 (TLR3) ligand Poly(I:C) has been shown to induce a viral aggravation of severe asthma by identifying double-stranded RNA (dsRNA). This study aimed to evaluate the therapeutic role of the TLR3/dsRNA complex inhibitor-calbiochem compound in the treatment of Poly(I:C)-induced viral asthma exacerbations through the ovalbu-min-induced asthma model in Swiss albino mice. Methods: Poly(I:C) and Ovalbumin drugs were injected in mice to sensitize (i.p. on 0, 7, and 14th day) and challenge (i.n. on the 21st and 22nd days). In contrast, the treatment drug TLR3/dsRNA complex inhibitor-calbiochem was given on the 21st and 22nd days intraperitoneally within the study period. In-vivo measurements were carried out in BALF and serum for pro-inflammatory cytokines, inflammatory leukocyte counts, lactate dehydrogenase (LDH) and nitrite levels, lungs/body weight index, and lung tissue histopathology using H and E staining in mice airways. Results: High levels of cytokines (NF-κB, IL-1β, IL-5, RANTES, MIP-2, and MCP-1) are seen in groups exposed to OVA and Poly (I:C). Further, inflammatory leukocyte cell counts, lung-body weight (LW/BW) index, airway hyperresponsiveness (AHR), and lung tissue damage sug-gest exacerbations in mice airways. On the other hand, TLR3/dsRNA complex inhibitor-calbio-chem and dexamethasone significantly reversed these changes toward normal levels. Conclusions: These results suggest that the novel compound TLR3/dsRNA complex inhibitor-calbiochem has a better therapeutic role than dexamethasone for managing inflammatory char-acteristics in asthmatic mice lungs and is a potent target for viral asthma exacerbations

Ism1 deficiency in mice exacerbates bleomycin-induced pulmonary fibrosis with enhanced cellular senescence and delayed fibrosis resolution

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing interstitial lung disease with high morbidity and mortality but unclear etiology and incomplete pathophysiological understandings, making the discovery of effective therapeutics arduous. Currently, two drugs, Nintedanib and Pirfenidone, are available for IPF treatment which can slow down the fibrotic scarring in the lung but are unable to provide disease resolution. Hence, further revelation of the molecular mechanisms of IPF is critical for the development of novel therapeutics. Isthmin-1 (ISM1) is a secreted anti-inflammatory protein highly expressed in the mouse and human lung. Ism1−/− mice presented spontaneous and progressive lung emphysema, as well as heightened acute lung injury (ALI) upon lipopolysaccharide (LPS) treatment with an accompanied increase of post-LPS-ALI pulmonary fibrosis. ISM1 is important for lung homeostasis with airway-delivered recombinant ISM1 (rISM1) suppressing cigarette smoke-induced emphysema, LPS-ALI, and house-dust mites (HDM)-induced asthma-like symptoms in mice. However, the role of ISM1 in pulmonary fibrosis is yet to be clearly understood. In this work, we show that Ism1−/− mice presented heightened bleomycin-induced pulmonary fibrosis (BIPF), with enhanced immune cell infiltration, myofibroblast accumulation, and collagen deposition. ISM1 deficiency also led to increased cellular senescence in mouse lungs, isolated primary alveolar type II epithelial cells, and primary lung fibroblasts upon bleomycin treatment. Ism1−/− mice also showed delayed resolution of pulmonary fibrosis with reduced lipofibroblasts and downregulation of lipid synthesis-related genes. These results are in congruence with the RNA-seq data which demonstrated gene expression alterations in Ism1−/− lung that are linked to predisposition to pulmonary fibrosis and dysregulation of lipid metabolism pathways. Gene expression analyses showed that Ism1 is similarly dysregulated in the lungs of BIPF and human IPF patients. These findings reveal an anti-fibrotic role of ISM1 in mouse lungs and provide the foundation to further investigate possible therapeutic applications of ISM1 for pulmonary fibrosis in the future.

A Technique to Generate and Deliver Drug Aerosols Directly to Mouse Lungs

A Technique to Generate and Deliver Drug Aerosols Directly to Mouse Lungs

A Technique to Generate and Deliver Drug Aerosols Directly to Mouse Lungs

A Technique to Generate and Deliver Drug Aerosols Directly to Mouse Lungs

IGATE analysis improves the interpretability of single-cell immune landscape of influenza infection.

Influenza poses a persistent health burden worldwide. To design equitable vaccines effective across all demographics, it is essential to better understand how host factors such as genetic background and aging affect the single-cell immune landscape of influenza infection. Cytometry by time-of-flight (CyTOF) represents a promising technique in this pursuit, but interpreting its large, high-dimensional data remains difficult. We have developed a new analytical approach, in silico gating annotating training elucidating (iGATE), based on probabilistic support vector machine classification. By rapidly and accurately "gating" tens of millions of cells in silico into user-defined types, iGATE enabled us to track 25 canonical immune cell types in mouse lung over the course of influenza infection. Applying iGATE to study effects of host genetic background, we show that the lower survival of C57BL/6 mice compared with BALB/c was associated with a more rapid accumulation of inflammatory cell types and decreased IL-10 expression. Furthermore, we demonstrate that the most prominent effect of aging is a defective T cell response, reducing survival of aged mice. Finally, iGATE reveals that the 25 canonical immune cell types exhibited differential influenza infection susceptibility and replication permissiveness in vivo, but neither property varied with host genotype or aging. The software is available at https://github.com/UmichWenLab/iGATE.