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High mobility group box 1 (HMGB1), a highly conserved nuclear protein, is released into the extracellular milieu serving as a modulator of inflammatory responses. HMGB1 levels are elevated in the airspaces of CF patients and mice with cystic fibrosis (CF)--like lung disease. To reduce extracellular release of HMGB1 into the lung airspaces, we previously introduced airway epithelial cell-specific HMGB1 deficiency in Scnn1b -Tg+ (Tg+) mice, a model of human CF-like lung disease. While the deletion of airway epithelial cell-specific HMGB1 did not reduce bronchoalveolar lavage fluid (BALF) HMGB1 levels, the intracellular HMGB1 deficiency resulted in exaggerated inflammatory responses. In this study, because HMGB1 protein levels were found significantly elevated in Tg+ BALF cells, we hypothesized that myeloid cell-derived HMGB1 deficiency will reduce extracellular HMGB1 levels in the lung airspaces of Tg+ mice, which will modulate pathological features of lung disease in Tg+ mice. Accordingly, we introduced myeloid cell-specific HMGB1 deficiency in Tg+ mice. The deletion of HMGB1 in myeloid cells did not result in any obvious alterations in the lungs of WT mice. The BALF levels of HMGB1 were comparable between myeloid cell-specific HMGB1-deficient Tg+ and HMGB1-sufficient Tg+ mice. As expected, as compared with the HMGB1-sufficient WT, HMGB1-sufficient Tg+ mice displayed increased total cell counts consisting of neutrophils, eosinophils, and lymphocytes. As compared with the HMGB1-sufficient Tg+ mice, the myeloid cell-specific HMGB1-deficient Tg+ mice exhibited significantly increased BALF neutrophil and eosinophil counts, which was associated with increased BALF levels of granulocyte-specific chemoattractants, i.e., KC/CXCL1, MCP-1/CCL2, MIP-1α/CCL3, and MIP-1β/CCL4, Th2 lymphocyte-specific chemoattractants, i.e., TARC/CCL17 and MDC/CCL22, and monocyte-specific chemoattractants, i.e., MCP-3/CCL7 and MCP-5/CCL12. As compared with the HMGB1-sufficient Tg+ mice, the alveolar macrophages from HMGB1-deficient Tg+ mice were significantly enlarged, suggesting their higher activation status. Myeloid cell-specific HMGB1 deletion also resulted in significantly improved clearance of spontaneous bacterial infection in Tg+ mice. Furthermore, myeloid cell-specific HMGB1 deletion significantly worsened pathological manifestations in Tg+ mice, i.e., exaggerated airway mucus obstruction, increased peribronchiolar infiltration of inflammatory cells, increased alveolar space enlargement, and increased lymphoid hyperplasia. Collectively, although myeloid cells are not the source of extracellular HMGB1 found in the inflamed airspaces of Tg+ mice, the myeloid cell-specific HMGB1 deficiency modulates key pathological features of mucoinflammatory lung disease in these mice.

Cre-LoxP system has been widely used to induce recombination of floxed genes of interest. Currently available macrophage promoter-specific Cre recombinase mice strains have various limitations that warrants the testing of additional Cre strains. V-maf m usculo a poneurotic f ibrosarcoma oncogene family, protein b - Cre ( Mafb-Cre ) mice label macrophages in most organs such as spleen, small intestine, lung, bone marrow, and peritoneal cavity. However, whether Mafb-Cre recombinase targets the gene recombination in alveolar macrophage remains untested. Here, we utilized Mafb Cre/WT R26 mTmG/WT strain that expresses mTOM protein in all the cells of mouse body except for those that express Mafb -Cre-regulated mEGFP. We performed fluorescent microscopy and flow cytometry to analyze mTOM and mEGFP expression in alveolar macrophages from Mafb Cre/WT R26 mTmG/WT mice. Our analyses revealed that the Mafb -Cre is active in only ~40% of the alveolar macrophages in an age-independent manner. While Mafb - (mTOM+/mEGFP-) and Mafb + (mEGFP+) alveolar macrophages exhibit comparable expression of CD11b and CD11c surface markers, the surface expression of MHCII is elevated in the Mafb + (mEGFP+) macrophages. The bone marrow-derived macrophages from Mafb Cre/WT R26 mTmG/WT mice are highly amenable to Cre-LoxP recombination in vitro. The bone marrow depletion and reconstitution experiment revealed that ~98% of alveolar macrophages from Mafb Cre/WT R26 mTmG/WT → WT chimera are amenable to the Mafb -Cre-mediated recombination. Finally, the Th2 stimulation and ozone exposure to the Mafb Cre/WT R26 mTmG/WT mice promote the Mafb -Cre-mediated recombination in alveolar macrophages. In conclusion, while the Mafb -/ Mafb + dichotomy thwarts the use of Mafb-Cre for the induction of floxed alleles in the entire alveolar macrophage population, this strain provides a unique tool to induce gene deletion in alveolar macrophages that encounter Th2 microenvironment in the lung airspaces.

High mobility group box 1 (HMGB1), a ubiquitous chromatin-binding protein required for gene transcription regulation, is released into the extracellular microenvironment by various structural and immune cells, where it is known to act as an alarmin. Here, we investigated the role of airway epithelium-specific HMGB1 in the pathogenesis of muco-obstructive lung disease in Scnn1b -transgenic (Tg+) mouse, a model of human cystic fibrosis (CF)-like lung disease. We hypothesized that airway epithelium-derived HMGB1 modulates muco-inflammatory lung responses in the Tg+ mice. The airway epithelium-specific HMGB1-deficient mice were generated and the effects of HMGB1 deletion on immune cell recruitment, airway epithelial cell composition, mucous cell metaplasia, and bacterial clearance were determined. The airway epithelium-specific deletion of HMGB1 in wild-type (WT) mice did not result in any morphological alterations in the airway epithelium. The deficiency of HMGB1 in airway epithelial cells in the Tg+ mice, however, resulted in significantly increased infiltration of macrophages, neutrophils, and eosinophils which was associated with significantly higher levels of inflammatory mediators, including G-CSF, KC, MIP-2, MCP-1, MIP-1α, MIP-1β, IP-10, and TNF-α in the airspaces. Furthermore, as compared to the HMGB1-sufficient Tg+ mice, the airway epithelial cell-specific HMGB1-deficient Tg+ mice exhibited poor resolution of spontaneous bacterial infection. The HMGB1 deficiency in the airway epithelial cells of Tg+ mice did not alter airway epithelial cell-specific responses including epithelial cell proliferation, mucous cell metaplasia, and mucus obstruction. Collectively, our findings provide novel insights into the role of airway epithelial cell-derived HMGB1 in the pathogenesis of CF-like lung disease in Tg+ mice.

Lung epithelial lining fluid (ELF) harbors a variety of proteins that influence homeostatic and stress responses in the airspaces. Exosomes, nano-sized extracellular vesicles, contain many proteins that vary in abundance and composition based on the prevailing conditions. Ozone causes inflammatory responses in the airspaces of experimental animals and humans. However, the exosomal protein signatures contained within the ELF from ozone-exposed lung airspaces remain poorly characterized. To explore this, we hypothesized that ozone triggers the release of exosome-bound inflammatory proteins from various cells that reflect mucoobstructive lung disease. Accordingly, we repetitively exposed adult male and female C57BL/6 mice to HEPA-filtered air (air) or 0.8 ppm ozone (4 h per day) for 14 days (five consecutive days of exposure, 2 days of rest, five consecutive days of exposure, 2 days of rest, four consecutive days of exposure). Exosome-bound proteomic signatures, as well as the levels of soluble inflammatory mediators in the bronchoalveolar lavage fluid (BALF), were determined 12–16 h after the last exposure. Principal component analyses of the exosome-bound proteome revealed a clear distinction between air-exposed and ozone-exposed mice, as well as between ozone-exposed males and ozone-exposed females. In addition to 575 proteins that were enriched in both sexes upon ozone exposure, 243 and 326 proteins were enriched uniquely in ozone-exposed males and females, respectively. Ingenuity pathway analyses on enriched proteins between ozone- and air-exposed mice revealed enrichment of pro-inflammatory pathways. More specifically, macrophage activation-related proteins were enriched in exosomes from ozone-exposed mice. Cytokine analyses on the BALF revealed elevated levels of G-CSF, KC, IP-10, IL-6, and IL-5 in ozone-exposed mice. Finally, the histopathological assessment revealed significantly enhanced intracellular localization of mucoinflammatory proteins including MUC5B and FIZZ1 in ozone-exposed mice in a cell-specific manner indicating the cellular sources of the proteins that are ferried in the exosomes upon ozone-induced lung injury. Collectively, this study identified exosomal, secretory, and cell-specific proteins and biological pathways following repetitive exposure of mice to ozone.

Exosomes, membrane-bound extracellular vesicles, ranging from approximately 30–200 nm in diameter, are released by almost all cell types and play critical roles in intercellular communication. In response to the prevailing stress, the exosome-bound protein signatures vary in abundance and composition. To identify the bronchoalveolar lavage fluid (BALF) exosome-bound proteins associated with mucoinflammatory lung disease and to gain insights into their functional implications, we compared BALF exosomes-derived proteins from adult Scnn1b transgenic ( Scnn1b -Tg+) and wild type (WT) mice. A total of 3,144 and 3,119 proteins were identified in BALF exosomes from Scnn1b -Tg+ and WT mice, respectively. Using cutoff criteria (Log 2 fold-change > 1 and adjusted p -value < 0.05), the comparison of identified proteins revealed 127 and 30 proteins that were significantly upregulated and downregulated, respectively, in Scnn1b -Tg+ versus WT mice. In addition, 52 and 27 proteins were exclusively enriched in Scnn1b -Tg+ and WT mice, respectively. The identified exosome-bound proteins from the homeostatic airspaces of WT mice were mostly relevant to the normal physiological processes. The protein signatures enriched in the BALF exosomes of Scnn1b -Tg+ mice were relevant to macrophage activation and mucoinflammatory processes. Ingenuity pathway analyses revealed that the enriched proteins in Scnn1b -Tg+ mice contributed to the inflammatory responses and antimicrobial defense pathways. Selective proteins including, RETNLA/FIZZ1, LGALS3/Galectin-3, S100A8/MRP8, and CHIL3/YM1 were immunolocalized to specific cell types. The comparative analysis between enriched BALF exosome proteins and previously identified differentially upregulated genes in Scnn1b -Tg+ versus WT mice suggested that the compartment-/cell-specific upregulation in gene expression dictates the enrichment of their respective proteins in the lung airspaces. Taken together, this study demonstrates that the BALF exosome-bound protein signatures reflect disease-relevant disturbances. Our findings suggest that the exosomes carry disease-relevant protein signatures that can be used as a diagnostic as well as predictive biomarkers for mucoinflammatory lung disease.

SARS-CoV-2, a novel coronavirus and an etiologic agent for the current global health emergency, causes acute infection of the respiratory tract leading to severe disease and significant mortality. Ever since the start of SARS-CoV-2, also known as the COVID-19 pandemic, countless uncertainties have been revolving around the pathogenesis and epidemiology of the SARS-CoV-2 infection. While air pollution has been shown to be strongly correlated to increased SARS-CoV-2 morbidity and mortality, whether environmental pollutants such as ground-level ozone affects the susceptibility of individuals to SARS-CoV-2 is not yet established. To investigate the impact of ozone inhalation on the expression levels of signatures associated with host susceptibility to SARS-CoV-2, we analyzed lung tissues collected from mice that were sub-chronically exposed to air or 0.8 ppm ozone for three weeks (4 h/night, 5 nights/week), and analyzed the expression of signatures associated with host susceptibility to SARS-CoV-2. SARS-CoV-2 entry into the host cells is dependent on the binding of the virus to the host cellular receptor, angiotensin-converting enzyme (ACE2), and its subsequent proteolytic priming by the host-derived protease, transmembrane protease serine 2 (TMPRSS2). The Ace2 transcripts were significantly elevated in the parenchyma, but not in the extrapulmonary airways and alveolar macrophages, from ozone-exposed mice. The TMPRSS2 protein and Tmprss2 transcripts were significantly elevated in the extrapulmonary airways, parenchyma, and alveolar macrophages from ozone-exposed mice. A significant proportion of additional known SARS-CoV-2 host susceptibility genes were upregulated in alveolar macrophages and parenchyma from ozone-exposed mice. Our data indicate that the unhealthy levels of ozone in the environment may predispose individuals to severe SARS-CoV-2 infection. Given the severity of this pandemic and the challenges associated with direct testing of host-environment interactions in clinical settings, we believe that this ozone exposure-based study informs the scientific community of the potentially detrimental effects of the ambient ozone levels in determining the host susceptibility to SARS-CoV-2.

Using zinc tellurium (ZnTe) as the buffer layer in the Cu 2 ZnSnS 4 (CZTS)-based solar cells showed an improvement in overall efficiency. ZnTe is investigated as an alternative to replace the conventional toxic Cd-contained buffer layers. It may also reduce the overall cost of these cells as both layers (ZnTe and CZTS) have eco-friendly and earth-abundant constituents. The sol–gel spin coating method is used for the deposition of CZTS thin films on the corning glass substrates. The X-ray diffraction studies showed the peaks corresponding to (112), (200), (220), and (312) planes which confirmed the formation of the essential kesterite phase. The optical band gap of the deposited films was found at around 1.45 eV by the UV–visible-NIR spectrophotometer. The optimum thickness of the absorber layer (CZTS) and buffer layer (ZnTe) was investigated based on the performance of the ZnO:Al/ZnO/ZnTe/CZTS/Mo cell structure by using the AMPS-1D simulation tool. In contrast, the tool was molded by the experimentally investigated data for the constituent materials of the cell structure. The solar cells’ efficiency was increased by 23.47% at 2500 nm and 50 nm thickness of the CZTS and ZnTe layers, respectively. In addition, it was analyzed and found that the current density value showed an improvement with operating temperature as it is one of the requirements in the high solar radiation areas where the temperature even rises more than 50 °C in the summer.

Mucoinflammatory lung disease in cystic fibrosis (CF) is characterized by airway surface liquid (ASL) layer dehydration and mucins hyperconcentration, which leads to airway obstruction, inflammation, bronchiectasis, and increased susceptibility to recurrent bacterial infections. Epidermal growth factor receptor (EGFR) is known to regulate airway mucous cell metaplasia (MCM) and mucins expression, but the role of EGFR pathway in the pathogenesis of CF-like lung disease remains unclear. Therefore, we hypothesized that airway epithelial cell-specific deficiency of EGFR mitigates mucoinflammatory responses in Scnn1b -transgenic (Tg+) mice that phenocopy human CF-like lung disease. To test this hypothesis, we examined the effect of airway epithelial cell-specific EGFR deficiency on the manifestation of mucoinflammatory outcomes in Tg+ mice. The airway epithelial cell-specific EGFR-deficient wild-type (WT) mice did not exhibit any obvious structural and functional defects in the lungs. The deletion of EGFR in airway epithelial cells in Tg+ mice, however, resulted in increased recruitment of neutrophils and macrophages into the lung airspaces, which was accompanied by significantly increased bronchoalveolar lavage fluid (BALF) levels of inflammatory mediators, including KC, G-CSF, MIP-2, MIP-1α, TNF-α, and MIP-1β. Additionally, as compared with the EGFR-sufficient Tg+ mice, the airway epithelial cell-specific EGFR-deficient Tg+ mice exhibited significantly increased postnatal mortality and compromised bacterial clearance. The deletion of EGFR in the airway epithelial cells of Tg+ mice resulted in an increased degree of mucus obstruction, which was associated with an increase in MCM and MUC5B production. Some of the molecular markers of type 2 inflammation, including Il13 , Slc26a4 , and Retnla , were significantly increased in airway epithelial cell-specific EGFR-deficient Tg+ mice versus EGFR-sufficient Tg+ mice. Taken together, our data show that EGFR deletion in the airway epithelial cells compromises postnatal survival, delays bacterial clearance, and modulates inflammatory and mucus obstruction-relevant endpoints, i.e., MCM, MUC5B production, and mucus obstruction, in Tg+ mice.

Germline deletion of certain genes causes embryonic lethality, therefore, understanding the effect of deletion of such genes on mammalian pathophysiology remains challenging. Tamoxifen (TAM)-inducible Cre recombinase is widely used for tissue-specific and temporal induction of gene deletion in mice. However, the tamoxifen treatment regimen for the generation of whole-body deletion of a gene is not yet fully standardized for the majority of organs/tissues. Accordingly, we employed GtROSA26 (R26) promoter-regulated Cre and a reporter gene expression strategy. GtROSA26 (R26) is an ubiquitous promoter and mice carrying the R26 Cre−ERT2 transgene express Cre-ERT2 in all the cells. Similarly, mice carrying the R26 mTOM-mEGFP transgene express mTOM (membrane-targeted tdTomato), in the absence of Cre or mEGFP (membrane-targeted enhanced green fluorescent protein), in the presence of Cre, in all the cells. The progeny carrying one allele of both transgenes were subjected to different TAM regimens, i.e., IP injections (4 injections; 1.35 mg/injection), diet (400 mg TAM-citrate/kg food), or diet (400 mg TAM-citrate/kg food) combined with either TAM-oral gavage (4 gavages; 1.35 mg/gavage) or TAM IP injections (4 injections; 1.35 mg/injection) for 2-weeks beginning at postnatal day (PND) 21 and the extent of Cre recombination in different tissues was determined at PND35. Tamoxifen administration resulted in a transient loss of body weight in all the treatment regimens with a relatively slower rate of weight gain in the TAM-diet plus TAM-oral gavage group compared to other groups. While the efficiency of Cre recombination, as determined by the expression of mEGFP protein, was variable among tissues, major tissues such as the liver, heart, lungs, spleen, and thymus—showed almost complete recombination. No recombination was evident in any of the tissues examined from the control mice. In general, the efficiency of Cre recombination was better with a combined regimen of TAM-diet with either TAM-injections or TAM-oral gavage compared to TAM-diet alone or TAM-injections alone. Our results demonstrate that a combination of TAM-diet with either TAM-injections or TAM-oral gavage can be employed for the efficient deletion of a gene in the whole body. Our findings will provide technical expertise to the researchers employing TAM-inducible Cre for the deletion of floxed genes in varied tissues.

Using an integrated strategy combining network pharmacology, molecular dynamics simulations, and in vivo experiments, this study explores the stage-specific targets of Chaihu Guizhi Decoction (CGD) for the treatment of Crohn's disease (CD). First, through the GEO database, the patients with Crohn's disease were divided into four groups: early patients (NCD), late patients (LCD), patients with postoperative recurrence (PCD) and patients without postoperative recurrence (UPCD), and the differential genes of each group were screened. Active ingredients in Chaihu Guizhi Decoction (CGD) with an oral bioavailability (OB) greater than 30% and drug-likeness (DL) exceeding 0.18 were screened using the TCMSP database, along with their corresponding potential targets. The target of CGD was intersected with four groups of differential genes. The common therapeutic targets of CGD for Crohn's disease in four groups and the specific therapeutic targets of each group were obtained. Core targets were identified through protein-protein interaction (PPI) network analysis. The potential mechanism of CGD in treating Crohn's disease was analyzed by enrichment of KEGG and GO. Finally, molecular docking and molecular dynamics simulation were used to verify the possibility of combining the effective ingredients of CGD with the therapeutic target of Crohn's disease. Construct a mouse model of inflammatory bowel disease and administer drug treatment. Use mouse intestinal tissue for Elisa, HE staining, and AB-PAS staining to validate the potential of drug therapy for inflammatory bowel disease. Meanwhile, Western blot experiments were conducted to verify the effect of quercetin on the expression of downstream target proteins. Through molecular docking verification, quercetin can bind to these six core genes. We analyzed quercetin and six gene protein products through molecular dynamics simulation. We found that the complex of quercetin and PTGS2 protein is relatively stable, which may be a therapeutic target. In animal studies, both CGD and quercetin significantly alleviated inflammation-induced intestinal shortening (p < 0.05). ELISA results demonstrated that CGD and quercetin markedly reduced the expression of pro-inflammatory cytokines, including IL-6, IL-1β, and TNF-α (p < 0.05). Furthermore, histopathological examinations via HE and PAS staining revealed a substantial mitigation of intestinal inflammation following treatment with CGD and quercetin. This study found through animal experiments that CGD and quercetin can treat CD. Through network pharmacology, molecular dynamics simulations, and Western blot experiments, it was demonstrated that quercetin can treat inflammatory bowel disease by affecting the expression of PTGS2 and IL-1B proteins.