Adult Mouse Lung Research Articles

Interferon lambda signaling in neutrophils enhances the pathogenesis of Bordetella pertussis infection

Abstract Interferon lambda plays diverse roles in bacterial infections. Previously, we showed that interferon lambda is induced in the lungs of Bordetella pertussis-infected adult mice and exacerbates inflammation. Here, we report that mice lacking the interferon lambda receptor 1 specifically on neutrophils (MRP8creIFNLR1fl/fl mice) exhibit reduced lung bacterial load and inflammation compared to wild-type mice during B. pertussis infection. In B. pertussis-infected wild-type mice, lung type I and III IFN responses were higher than in MRP8creIFNLR1fl/fl mice, correlating with increased lung inflammatory pathology. There was an increased proportion of interferon gamma-producing neutrophils in the lungs of MRP8creIFNLR1fl/fl mice compared to wild-type mice. IFNLR1−/− neutrophils incubated with B. pertussis exhibited higher killing compared to wild-type neutrophils. Treatment of wild-type neutrophils with interferon lambda further decreased their bacterial killing capacity and treatment of wild-type mice with interferon lambda increased lung bacterial loads. Contributing to the differential killing, we found that IFNLR1−/− neutrophils exhibit higher levels of reactive oxygen species, myeloperoxidase, matrix metalloproteinase-9 activity, neutrophil extracellular traps, and interferon gamma secretion than wild-type neutrophils, and inhibiting NADPH oxidase inhibited bacterial killing in IFNLR1−/− neutrophils. B. pertussis-induced interferon lambda secretion and IFNLR1 gene expression in mouse and human neutrophils and this was dependent on the bacterial virulence protein pertussis toxin. Pertussis toxin enhanced bacterial loads in wild type but not in MRP8creIFNLR1fl/fl or IFNLR1−/− mice. Thus, pertussis toxin disrupts neutrophil function by enhancing type III IFN signaling, which prevents neutrophils from effectively clearing B. pertussis during infection, leading to higher bacterial loads and exacerbation of lung inflammation.

Influenza induces lung lymphangiogenesis independent of YAP/TAZ activity in lymphatic endothelial cells

The lymphatic system consists of a vessel network lined by specialized lymphatic endothelial cells (LECs) that are responsible for tissue fluid homeostasis and immune cell trafficking. The mechanisms for organ-specific LEC responses to environmental cues are not well understood. We found robust lymphangiogenesis during influenza A virus infection in the adult mouse lung. We show that the number of LECs increases twofold at 7 days post-influenza infection (dpi) and threefold at 21 dpi, and that lymphangiogenesis is preceded by lymphatic dilation. We also show that the expanded lymphatic network enhances fluid drainage to mediastinal lymph nodes. Using EdU labeling, we found that a significantly higher number of pulmonary LECs are proliferating at 7 dpi compared to LECs in homeostatic conditions. Lineage tracing during influenza indicates that new pulmonary LECs are derived from preexisting LECs rather than non-LEC progenitors. Lastly, using a conditional LEC-specific YAP/TAZ knockout model, we established that lymphangiogenesis, fluid transport and the immune response to influenza are independent of YAP/TAZ activity in LECs. These findings were unexpected, as they indicate that YAP/TAZ signaling is not crucial for these processes.

Impact of aging and ergothioneine pre-treatment on naphthalene toxicity in lung

Aging increases susceptibility to lung disease, but the topic is understudied, especially in relation to environmental exposures with the bulk of rodent studies using young adults. This study aims to define the pulmonary toxicity of naphthalene (NA) and the impacts of a dietary antioxidant, ergothioneine (ET), in the liver and lungs of middle-aged mice. NA causes a well-characterized pattern of conducting airway epithelial injury in the lung in young adult mice, but NA’s toxicity has not been characterized in middle-aged mice, aged 1–1.5 years. ET is a dietary antioxidant that is synthesized by bacteria and fungi. The ET transporter (ETT), SLC22A4, is upregulated in tissues that experience high levels of oxidative stress. In this study, middle-aged male and female C57BL/6 J mice, maintained on an ET-free synthetic diet from conception, were gavaged with 70 mg/kg of ET for five consecutive days. On day 8, the mice were exposed to a single intraperitoneal NA dose of 50, 100, 150, or 200 mg/kg. At 24 hours post NA injection samples were collected and analyzed for ET concentration and reduced (GSH) and oxidized glutathione (GSSG) concentrations. Histopathology, morphometry, and gene expression were examined. Histopathology of mice exposed to 100 mg/kg of NA suggests reduction in toxicity in the terminal airways of both male (p ≤ 0.001) and female (p ≤ 0.05) middle-aged mice by the ET pretreatment. Our findings in this study are the first to document the toxicity of NA in middle-aged mice and show some efficacy of ET in reducing NA toxicity.

Impact of different tissue dissociation protocols on endothelial cell recovery from developing mouse lungs.

Flow cytometry and fluorescence-activated cell sorting are widely used to study endothelial cells, for which the generation of viable single-cell suspensions is an essential first step. Two enzymatic approaches, collagenase A and dispase, are widely employed for endothelial cell isolation. In this study, the utility of both enzymatic approaches, alone and in combination, for endothelial cell isolation from juvenile and adult mouse lungs was assessed, considering the number, viability, and subtype composition of recovered endothelial cell pools. Collagenase A yielded an 8-12-fold superior recovery of viable endothelial cells from lung tissue from developing mouse pups, compared to dispase, although dispase proved superior in efficiency for epithelial cell recovery. Single-cell RNA-Seq revealed that the collagenase A approach yielded a diverse endothelial cell subtype composition of recovered endothelial cell pools, with broad representation of arterial, capillary, venous, and lymphatic lung endothelial cells; while the dispase approach yielded a recovered endothelial cell pool highly enriched for one subset of general capillary endothelial cells, but poor representation of other endothelial cells subtypes. These data indicate that tissue dissociation markedly influences the recovery of endothelial cells, and the endothelial subtype composition of recovered endothelial cell pools, as assessed by single-cell RNA-Seq.

Genetically-engineered mouse models of small cell lung cancer: the next generation.

Small cell lung cancer (SCLC) remains the most fatal form of lung cancer, with patients in dire need of new and effective therapeutic approaches. Modeling SCLC in an immunocompetent host is essential for understanding SCLC pathogenesis and ultimately discovering and testing new experimental therapeutic strategies. Human SCLC is characterized by near universal genetic loss of the RB1 and TP53 tumor suppressor genes. Twenty years ago, the first genetically-engineered mouse model (GEMM) of SCLC was generated using conditional deletion of both Rb1 and Trp53 in the lungs of adult mice. Since then, several other GEMMs of SCLC have been developed coupling genomic alterations found in human SCLC with Rb1 and Trp53 deletion. Here we summarize how GEMMs of SCLC have contributed significantly to our understanding of the disease in the past two decades. We also review recent advances in modeling SCLC in mice that allow investigators to bypass limitations of the previous generation of GEMMs while studying new genes of interest in SCLC. In particular, CRISPR/Cas9-mediated somatic gene editing can accelerate how new genes of interest are functionally interrogated in SCLC tumorigenesis. Notably, the development of allograft models and precancerous precursor models from SCLC GEMMs provides complementary approaches to GEMMs to study tumor cell-immune microenvironment interactions and test new therapeutic strategies to enhance response to immunotherapy. Ultimately, the new generation of SCLC models can accelerate research and help develop new therapeutic strategies for SCLC.

Activation of mTOR signaling in adult lung microvascular progenitor cells accelerates lung aging.

Reactivation and dysregulation of the mTOR signaling pathway are a hallmark of aging and chronic lung disease; however, the impact on microvascular progenitor cells (MVPCs), capillary angiostasis, and tissue homeostasis is unknown. While the existence of an adult lung vascular progenitor has long been hypothesized, these studies show that Abcg2 enriches for a population of angiogenic tissue-resident MVPCs present in both adult mouse and human lungs using functional, lineage, and transcriptomic analyses. These studies link human and mouse MVPC-specific mTORC1 activation to decreased stemness, angiogenic potential, and disruption of p53 and Wnt pathways, with consequent loss of alveolar-capillary structure and function. Following mTOR activation, these MVPCs adapt a unique transcriptome signature and emerge as a venous subpopulation in the angiodiverse microvascular endothelial subclusters. Thus, our findings support a significant role for mTOR in the maintenance of MVPC function and microvascular niche homeostasis as well as a cell-based mechanism driving loss of tissue structure underlying lung aging and the development of emphysema.

Lung Megakaryocytes Arise from a Flt3 - Cell Lineage and Contribute to Recovery of Platelet Counts in Thrombocytopenic States

Platelet-producing megakaryocytes (Mks) are present in the lung, and we have described an immune modulatory phenotype of lung Mks. It is assumed that the extravascular lung Mks are ‘seeded’ from the bone marrow (BM). To investigate lung Mk origin and how origin may impact function, we used complementary models to lineage trace lung Mks and their platelets. FlkSwitch mTmG mice label all progeny from Flt3 + progenitors with GFP while all other cells are labeled with Tomato. We leveraged the FlkSwitch mouse model to compare BM and lung Mk origins. In 18 week old mice, the vast majority of BM and spleen Mks were GFP +, while lung Mks were largely Tomato + (Figure 1A). These data implied that in the adult mouse lung Mks arise from a Flt3 - pathway that is distinct from BM megakaryopoiesis in homeostatic conditions. We next used a lineage tracing model (MDS1-Cre-ERT2 Tomato) where tamoxifen (TAM) administration induces a Tomato reporter in hematopoietic stem cell (HSC)-derived cells. Mice were dosed with TAM at 18 weeks and sacrificed at 32 weeks. Lung and BM Mks were largely Tomato + (~75%) and matched Tomato levels of BM Lineage - Sca1 + c-Kit + (LSK). Next, we dosed mice with TAM at 8 weeks, sacrificed the mice at 14 weeks and found similarly high levels of Tomato in BM LSK, BM Mk, and lung Mk. In MDS1-Cre-ERT2 Tomato mice, both BM and lung Mks were Tomato +/HSC-dependent, suggesting that lung Mks arise from HSCs in adult mice. Next, we used CFSE dye given oropharyngeal (OP) to label only lung cells. We detected CFSE + lung Mks for up to 120 days post administration without any CFSE + Mks in BM or spleen. These data suggested there is a long-lived Mk population in the lung. Taken together, these complementary models imply that lung Mks arise from a Flt3 - lineage that is HSC-dependent, which yields Mks that can live in the lung for extended periods of time. The CFSE OP method labeled only lung cells, which enabled us to track lung Mk-derived platelets. We found that about 10% of the steady-state circulating platelet pool is lung derived. In a model of thrombocytopenia using diphtheria toxin mediated platelet depletion in PF4-Cre-iDTR mice, we observed a doubling of CFSE + lung-derived circulating platelets (about 20%). We also used Plasmodium yoelli murine malaria as a model of infection associated with more chronic thrombocytopenia. Using CFSE OP labeling of lung Mk-derived platelets, we saw a marked increase in CFSE + lung-derived platelets early post- P. Yoelli infection (Figure 1B). Our studies suggest that lung Mks arise from a Flt3 - HSC-dependent pathway and contribute relatively more platelets during acute and chronic thrombocytopenia. Ongoing work aims to describe the role of lung megakaryopoiesis via a Flt3 - HSC-dependent pathway in early development and in models of thrombocytopenia.

Vascularization of the adult mouse lung grafted onto the chick chorioallantoic membrane

In the later stages of angiogenesis, the vascular sprout transitions into a functional vessel by fusing with a target vessel. Although this process appears to routinely occur in embryonic tissue, the biologic rules for sprout fusion and lumenization in adult regenerating tissue are unknown. To investigate this process, we grafted portions of the regenerating post-pneumonectomy lung onto the chick chorioallantoic membrane (CAM). Grafts from all 4 lobes of the post-pneumonectomy right lung demonstrated peri-graft angiogenesis as reflected by fluorescent plasma markers; however, fluorescent microsphere perfusion primarily occurred in the lobe of the lung that is the dominant site of post-pneumonectomy angiogenesis—namely, the cardiac lobe. Vascularization of the cardiac lobe grafts was confirmed by active tissue growth (p < .05). Functional vascular connections between the cardiac lobe and the CAM vascular network were demonstrated by confocal fluorescence microscopy as well as corrosion casting and scanning electron microscopy (SEM). Bulk transcriptional profiling of the cardiac lobe demonstrated the enhanced expression of many genes relative to alveolar epithelial cell (CD11b−/CD31−) control cells, but only the upregulation of Ereg and Fgf6 compared to the less well-vascularized right upper lobe. The growth of actively regenerating non-neoplastic adult tissue on the CAM demonstrates that functional lumenization can occur between species (mouse and chick) and across the developmental spectrum (adult and embryo).

Flk1 Deficiency and Hypoxia Synergistically Promote Endothelial Dysfunction, Vascular Remodeling, and Pulmonary Hypertension.

The mechanisms underlying pulmonary hypertension (PH) remain largely unknown; further, why advanced vascular remodeling preferentially occurs in arterioles is yet to be answered. VEGF (vascular endothelial growth factor) regulates angiogenesis through Flk1 and Flt1 on endothelial cells (ECs), which may be related to PH pathogenesis. However, spatiotemporal expression patterns of Flk1 and Flt1 in the pulmonary vascular system and the role of endothelial Flk1 in PH development remain poorly understood. We analyzed multiple reporter mice, including Flk1-GFP bacterial artificial chromosome transgenic (Tg), Flt1-DsRed bacterial artificial chromosome Tg, and Flk1-GFP/Flt1-DsRed double Tg mice, to determine the spatiotemporal expression of Flk1 and Flt1 in hypoxia-induced PH. We also used Cdh5CreERT2/Flk1f/f/Tomato (Flk1-KO) mice to induce EC-specific Flk1 deletion and lineage tracing in chronic hypoxia. Flk1 was specifically expressed in the ECs of small pulmonary vessels, including arterioles. Conversely, Flt1 was more broadly expressed in the ECs of large- to small-sized vessels in adult mouse lungs. Intriguingly, Flk1+ ECs were transiently increased in hypoxia with proliferation, whereas Flt1 expression was unchanged. Flk1-KO mice did not exhibit pulmonary vascular remodeling nor PH in normoxia; however, the arteriolar ECs changed to a cuboidal shape with protrusion. In hypoxia, Flk1 deletion exacerbated EC dysfunction and reduced their number via apoptosis. Additionally, Flk1 deletion promoted medial thickening and neointimal formation in arterioles and worsened PH. Mechanistically, lineage tracing revealed that neointimal cells were derived from Flk1-KO ECs. Moreover, RNA sequencing in pulmonary ECs demonstrated that Flk1 deletion and hypoxia synergistically activated multiple pathways, including cell cycle, senescence/apoptosis, and cytokine/growth factor, concomitant with suppression of cell adhesion and angiogenesis, to promote vascular remodeling. Flk1 and Flt1 were differentially expressed in pulmonary ECs. Flk1 deficiency and hypoxia jointly dysregulated arteriolar ECs to promote vascular remodeling. Thus, dysfunction of Flk1+ ECs may contribute to the pathogenesis of advanced vascular remodeling in pulmonary arterioles.

Effects of a lycopene-layered double hydroxide composite administration in cells and lungs of adult mice.

Lycopene is a natural compound with one of the highest antioxidant activities. Its consumption is associated with lower risks in lung cancer and chronic obstructive pulmonary disease, for example. Experimentally, a murine model demonstrated the ingestion of lycopene, which reduced the damage in lungs caused by cigarette smoke. Since lycopene is highly hydrophobic, its formulations in supplements and preparations for laboratory assays are based on oils, additionally, bioavailavility is low. We developed a lycopene layered double hydroxide (Lyc-LDH) composite, which is capable of transporting lycopene aqueous media. Our objective was to evaluate the cytotoxicity of Lyc-LDH and the intra-cellular production of reactive oxygen species (ROS) in J774A.1 cells. Also, in vivo assays were conducted with 50 male C57BL/6 mice intranasally treated with Lyc-LDH 10mg/kg (LG10), Lyc-LDH 25mg/kg (LG25) and Lyc-LDH 50mg/kg (LG50) during five days compared against a vehicle (VG) and control (CG) group. The blood, bronchoalveolar lavage fluid (BALF) and lung tissue were analyzed. The results revealed that Lyc-LDH composite attenuated intracellular ROS production stimulated with lipopolysacharide. In BALF, the highest doses of Lyc-LDH (LG25 and LG50) promoted influx of macrophages, lymphocytes, neutrophils and eosinophils compared to CG and VG. Also, LG50 increased the levels of IL-6 and IL-13, and promoted the redox imbalance in the pulmonary tissue. On the contrary, low concentrations did not produce significative effects. In conclusion, our results suggest that intranasal administration of high concentrations of Lyc-LDH induces inflammation as well as redox status changes in the lungs of healthy mice, however, results with low concentrations open a promising way to study LDH composites as vehicles for intranasal administration of antioxidant coadjuvants.

Intrauterine exposure to nicotine through maternal vaping disrupts embryonic lung and skeletal development via the Kcnj2 potassium channel

Smoking cigarettes during pregnancy is associated with adverse effects on infants including low birth weight, defective lung development, and skeletal abnormalities. Pregnant women are increasingly turning to vaping [use of electronic (e)-cigarettes] as a perceived safer alternative to cigarettes. However, nicotine disrupts fetal development, suggesting that like cigarette smoking, nicotine vaping may be detrimental to the fetus. To test the impact of maternal vaping on fetal lung and skeletal development in mice, pregnant dams were exposed to e-cigarette vapor throughout gestation. At embryonic day (E)18.5, vape exposed litter sizes were reduced, and some embryos exhibited growth restriction compared to air exposed controls. Fetal lungs were collected for histology and whole transcriptome sequencing. Maternally nicotine vaped embryos exhibited histological and transcriptional changes consistent with impaired distal lung development. Embryonic lung gene expression changes mimicked transcriptional changes observed in adult mouse lungs exposed to cigarette smoke, suggesting that the developmental defects may be due to direct nicotine exposure. Fetal skeletons were analyzed for craniofacial and long bone lengths. Nicotine directly binds and inhibits the Kcnj2 potassium channel which is important for bone development. The length of the maxilla, palatal shelves, humerus, and femur were reduced in vaped embryos, which was further exacerbated by loss of one copy of the Kcnj2 gene. Nicotine vapor exposed Kcnj2KO/+ embryos also had significantly lower birth weights than unexposed animals of either genotype. Kcnj2 mutants had severely defective lungs with and without vape exposure, suggesting that potassium channels may be broadly involved in mediating the detrimental developmental effects of nicotine vaping. These data indicate that intrauterine nicotine exposure disrupts fetal lung and skeletal development likely through inhibition of Kcnj2.

Loss of cytochrome P450 (CYP)1B1 mitigates hyperoxia response in adult mouse lung by reprogramming metabolism and translation

Oxygen supplementation is life saving for premature infants and for COVID-19 patients but can induce long-term pulmonary injury by triggering inflammation, with xenobiotic-metabolizing CYP enzymes playing a critical role. Murine studies showed that CYP1B1 enhances, while CYP1A1 and CYP1A2 protect from, hyperoxic lung injury. In this study we tested the hypothesis that Cyp1b1-null mice would revert hyperoxia-induced transcriptomic changes observed in WT mice at the transcript and pathway level. Wild type (WT) C57BL/6J and Cyp1b1-null mice aged 8–10 weeks were maintained in room air (21% O2) or exposed to hyperoxia (>95% O2) for 48h. Transcriptomic profiling was conducted using the Illumina microarray platform. Hyperoxia exposure led to robust changes in gene expression and in the same direction in WT, Cyp1a1-, Cyp1a2-, and Cyp1b1-null mice, but to different extents for each mouse genotype. At the transcriptome level, all Cyp1-null murine models reversed hyperoxia effects. Gene Set Enrichment Analysis identified 118 hyperoxia-affected pathways mitigated only in Cyp1b1-null mice, including lipid, glutamate, and amino acid metabolism. Cell cycle genes Cdkn1a and Ccnd1 were induced by hyperoxia in both WT and Cyp1b1-null mice but mitigated in Cyp1b1-null O2 compared to WT O2 mice. Hyperoxia gene signatures associated positively with bronchopulmonary dysplasia (BPD), which occurs in premature infants (with supplemental oxygen being one of the risk factors), but only in the Cyp1b1-null mice did the gene profile after hyperoxia exposure show a partial rescue of BPD-associated transcriptome. Our study suggests that CYP1B1 plays a pro-oxidant role in hyperoxia-induced lung injury.

Efficient Adeno-associated Virus-mediated Transgenesis in Alveolar Stem Cells and Associated Niches.

Targeted delivery of transgenes to tissue-resident stem cells and related niches offers avenues for interrogating pathways and editing endogenous alleles for therapeutic interventions. Here, we survey multiple adeno-associated virus (AAV) serotypes, administered via intranasal and retroorbital routes in mice, to target lung alveolar stem cell niches. We found that AAV5, AAV4, and AAV8 efficiently and preferentially transduce alveolar type-2 stem cells (AT2s), endothelial cells, and PDGFRA+ fibroblasts, respectively. Notably, some AAVs show different cell tropisms depending on the route of administration. Proof-of-concept experiments reveal the versatility of AAV5-mediated transgenesis for AT2-lineage labeling, clonal cell tracing after cell ablation, and conditional gene inactivation in both postnatal and adult mouse lungs invivo. AAV6, but not AAV5, efficiently transduces both mouse and human AT2s in alveolar organoid cultures. Furthermore, AAV5 and AAV6 can be used to deliver guide RNAs and transgene cassettes for homologous recombination invivo and ex vivo, respectively. Using this system coupled with clonal derivation of AT2 organoids, we demonstrate efficient and simultaneous editing of multiple loci, including targeted insertion of a payload cassette in AT2s. Taken together, our studies highlight the powerful utility of AAVs for interrogating alveolar stem cells and other specific cell types both invivo and ex vivo.

Acute and Repeated Ozone Exposures Differentially Affect Circadian Clock Gene Expression in Mice.

Circadian rhythms have an established role in regulating physiological processes, such as inflammation, immunity, and metabolism. Ozone, a common environmental pollutant with strong oxidative potential, is implicated in lung inflammation/injury in asthmatics. However, whether O3 exposure affects the expression of circadian clock genes in the lungs is not known. In this study, changes in the expression of core clock genes are analyzed in the lungs of adult female and male mice exposed to filtered air (FA) or O3 using qRT-PCR. The findings are confirmed using an existing RNA-sequencing dataset from repeated FA- and O3 -exposed mouse lungs and validated by qRT-PCR. Acute O3 exposure significantly alters the expression of clock genes in the lungs of females (Per1, Cry1, and Rora) and males (Per1). RNA-seq data revealing sex-based differences in clock gene expression in the airway of males (decreased Nr1d1/Rev-erbα) and females (increased Skp1), parenchyma of females and males (decreased Nr1d1 and Fbxl3 and increased Bhlhe40 and Skp1), and alveolar macrophages of males (decreased Arntl/Bmal1, Per1, Per2, Prkab1, and Prkab2) and females (increased Cry2, Per1, Per2, Csnk1d, Csnk1e, Prkab2, and Fbxl3). These findings suggest that lung inflammation caused by O3 exposure affects clock genes which may regulate key signaling pathways.

Identification of a subset of lung neutrophils that promote Acute Lung Injury resolution in mice

Abstract The acute respiratory distress syndrome (ARDS) is a major cause of respiratory failure with limited therapeutic options. Unresolving inflammation in the lungs and alveolar-capillary barrier dysfunction are main features of ARDS. The sharp increase in the incidence of ARDS during COVID19 emphasized the need for a better understanding of immunological events in ARDS that inform novel therapeutics. Here, we used a model of self-limited acid-induced ALI by selectively instilling HCL to the left lung of adult mice. The time-course of barrier dysfunction was determined via quantification of lung consolidation by micro-computed tomography, while inflammation was assessed by histology and flow cytometry. Neutrophils, rapidly recruited into the lungs, decreased over time during resolution while macrophage numbers, reduced during initial stages, increased. Immunophenotyping of the leukocyte recruitment to the lungs during the resolution of injury uncovered a neutrophils’ subset that was expressing SiglecF. These cells were distinct from eosinophils and phenotypically, and functionally different from the classical SiglecF-negative neutrophils. The roles for SiglecF+ neutrophils were further assessed by in vitro functional studies, adoptive transfer experiments, and lipidomic analysis. Depletion of neutrophils impaired alveolar epithelial cell type II (AT2) repair, while the adoptive transfer of SiglecF+ neutrophils accelerated epithelial cell restitution. Among the mechanisms associated with the SiglecF+ neutrophils in lung repair was the production of specialized pro-resolving mediators and CSF1. In sum, we identified protective roles for a subset of tissue neutrophils during the resolution of lung injury in mice.

Galectin-9 promotes natural killer cells activity via interaction with CD44.

Natural killer (NK) cells are a potent innate source of cytokines and cytoplasmic granules. Their effector functions are tightly synchronized by the balance between the stimulatory and inhibitory receptors. Here, we quantified the proportion of NK cells and the surface presence of Galectin-9 (Gal-9) from the bone marrow, blood, liver, spleen, and lungs of adult and neonatal mice. We also examined the effector functions of Gal-9+NK cells compared with their Gal-9- counterparts. Our results revealed that Gal-9+NK cells are more abundant in tissues, in particular, in the liver than in the blood and bone marrow. We found Gal-9 presence was associated with enhanced cytotoxic effector molecules granzyme B (GzmB) and perforin expression. Likewise, Gal-9 expressing NK cells displayed greater IFN-γ and TNF-α expression than their negative counterparts under hemostatic circumstances. Notably, the expansion of Gal-9+NK cells in the spleen of mice infected with E. coli implies that Gal-9+NK cells may provide a protective role against infection. Similarly, we found the expansion of Gal-9+NK cells in the spleen and tumor tissues of melanoma B16-F10 mice. Mechanistically, our results revealed the interaction of Gal-9 with CD44 as noted by their co-expression/co-localization. Subsequently, this interaction resulted in enhanced expression of Phospho-LCK, ERK, Akt, MAPK, and mTOR in NK cells. Moreover, we found Gal-9+NK cells exhibited an activated phenotype as evidenced by increased CD69, CD25, and Sca-1 but reduced KLRG1 expression. Likewise, we found Gal-9 preferentially interacts with CD44high in human NK cells. Despite this interaction, we noted a dichotomy in terms of effector functions in NK cells from COVID-19 patients. We observed that the presence of Gal-9 on NK cells resulted in a greater IFN-γ expression without any changes in cytolytic molecule expression in these patients. These observations suggest differences in Gal-9+NK cell effector functions between mice and humans that should be considered in different physiological and pathological conditions. Therefore, our results highlight the important role of Gal-9 via CD44 in NK cell activation, which suggests Gal-9 is a potential new avenue for the development of therapeutic approaches to modulate NK cell effector functions.

Combination of pioglitazone, a PPARγ agonist, and synthetic surfactant B-YL prevents hyperoxia-induced lung injury in adult mice lung explants

IntroductionHyperoxia-induced lung injury is characterized by acute alveolar injury, disrupted epithelial-mesenchymal signaling, oxidative stress, and surfactant dysfunction, yet currently, there is no effective treatment. Although a combination of aerosolized pioglitazone (PGZ) and a synthetic lung surfactant (B-YL peptide, a surfactant protein B mimic) prevents hyperoxia-induced neonatal rat lung injury, whether it is also effective in preventing hyperoxia-induced adult lung injury is unknown. MethodUsing adult mice lung explants, we characterize the effects of 24 and 72-h (h) exposure to hyperoxia on 1) perturbations in Wingless/Int (Wnt) and Transforming Growth Factor (TGF)-β signaling pathways, which are critical mediators of lung injury, 2) aberrations of lung homeostasis and injury repair pathways, and 3) whether these hyperoxia-induced aberrations can be blocked by concomitant treatment with PGZ and B-YL combination. ResultsOur study reveals that hyperoxia exposure to adult mouse lung explants causes activation of Wnt (upregulation of key Wnt signaling intermediates β-catenin and LEF-1) and TGF-β (upregulation of key TGF-β signaling intermediates TGF-β type I receptor (ALK5) and SMAD 3) signaling pathways accompanied by an upregulation of myogenic proteins (calponin and fibronectin) and inflammatory cytokines (IL-6, IL-1β, and TNFα), and alterations in key endothelial (VEGF-A and its receptor FLT-1, and PECAM-1) markers. All of these changes were largely mitigated by the PGZ + B-YL combination. ConclusionThe effectiveness of the PGZ + B-YL combination in blocking hyperoxia-induced adult mice lung injury ex-vivo is promising to be an effective therapeutic approach for adult lung injury in vivo.

Cnr1−/− has minimal impact on chlorpyrifos-mediated effects in the mouse endocannabinoid system, but it does alter lipopolysaccharide-induced cytokine levels in splenocytes

Chlorpyrifos (CPF) is an organophosphate pesticide that can inhibit endocannabinoid (eCB) metabolizing enzymes in animal models at levels that do not significantly alter acetylcholinesterase (AChE) in the central nervous system (CNS). Previous studies indicated that repeated low-level CPF exposure in developing rats increased the levels of eCBs in the brain. Because eCBs play a role in immune homeostasis through their engagement with cannabinoid receptors, we investigated the role of cannabinoid receptor 1 (CB1, encoded by the Cnr1 gene) on the CPF-mediated effects in the spleen and lung of neonatal and adult female mice. We treated neonatal and adult female Cnr1−/− mice with 2.5 mg/kg oral CPF or vehicle for 7 days. Tissues were harvested 4 h after the last CPF dose to evaluate eCB metabolic enzyme activity, levels of eCBs, and tissue immunophenotype. There were a small number of genotype-dependent alterations noted in the endpoints following CPF treatment that were specific to age and tissue type, and differences in eCB metabolism caused by CPF treatment did not correlate to changes in eCB levels. To explore the role of CB1 in CPF-mediated effects on immune endpoints, in vitro experiments were performed with WT murine splenocytes exposed to chlorpyrifos oxon (CPO; oxon metabolite of CPF) and challenged with lipopolysaccharide (LPS). While CPO did not alter LPS-induced pro-inflammatory cytokine levels, inactivation of CB1 by the antagonist SR141716A augmented LPS-induced IFN-γ levels. Additional experiments with WT and Cnr1−/− murine splenocytes confirmed a role for CB1 in altering the production of LPS-induced pro-inflammatory cytokine levels. We conclude that CPF-mediated effects on the eCB system are not strongly dependent on CB1, although abrogation of CB1 does alter LPS-induced cytokine levels in splenocytes.

Nephronectin is required to maintain right lung lobar separation during embryonic development.

Nephronectin (NPNT) is a basement membrane (BM) protein and high-affinity ligand of integrin α8β1 that is required for kidney morphogenesis in mice. In the lung, NPNT also localizes to BMs, but its potential role in pulmonary development has not been investigated. Mice with a floxed Npnt allele were used to generate global knockouts (KOs). Staged embryos were obtained by timed matings of heterozygotes and lungs were isolated for analysis. Although primary and secondary lung bud formation was normal in KO embryos, fusion of right lung lobes, primarily the medial and caudal, was first detected at E13.5 and persisted into adulthood. The lung parenchyma of KO mice was indistinguishable from wild-type (WT) and lobe fusion did not alter respiratory mechanics in adult KO mice. Interrogation of an existing single-cell RNA-seq atlas of embryonic and adult mouse lungs identified Npnt transcripts in mesothelial cells at E12.5 and into the early postnatal period, but not in adult lungs. KO embryonic lungs exhibited increased expression of laminin α5 and deposition of collagen IV in the mesothelial BM, accompanied by abnormalities in collagen fibrils in the adjacent stroma. Cranial and accessory lobes extracted from KO embryonic lungs fused ex vivo when cultured in juxtaposition, with the area of fusion showing loss of the mesothelial marker Wilms tumor 1. Because a similar pattern of lobe fusion was previously observed in integrin α8 KO embryos, our results suggest that NPNT signaling through integrin α8, likely in the visceral pleura, maintains right lung lobe separation during embryogenesis.

Characterization of a novel Hoxa5eGFP mouse line.

Hox genes encode transcription factors that are important for establishing the body plan. Hoxa5 is a member of the mammalian Hox5 paralogous group that regulates the patterning and morphology of the cervical-thoracic region of the axial skeleton. Hoxa5 also plays crucial functions in lung morphogenesis. We generated a Hoxa5eGFP reporter mouse line using CRISPR technology, allowing real-time visualization of Hoxa5 expression. Hoxa5eGFP recapitulates reported embryonic Hoxa5 mRNA expression patterns. Specifically, Hoxa5eGFP can be visualized in the developing mouse neural tube, somites, lung, diaphragm, foregut, and midgut, among other organs. In the stomach, posteriorly biased Hoxa5eGFP expression correlates with a drastic morphological reduction of the corpus in Hox5 paralogous mutants. Expression of Hoxa5eGFP in the lung continues in all lung fibroblast populations through postnatal and adult stages. We identified cell types that express Hoxa5 in postnatal and adult mouse lungs, including various fibroblasts and vascular endothelial cells. This reporter line will be a powerful tool for studies of the function of Hoxa5 during mouse development, homeostasis, and disease processes.