Lung Mononuclear Cells Research Articles

CompCorona: A web application for comparative transcriptome analyses of coronaviruses reveals SARS-CoV-2-specific host response.

Understanding the mechanism of host transcriptomic response to infection by the SARS-CoV-2 virus is crucial, especially for patients suffering from long-term effects of COVID-19, such as long COVID or pericarditis inflammation, potentially linked to side effects of the SARS-CoV-2 spike proteins. We conducted comprehensive transcriptome and enrichment analyses on lung and peripheral blood mononuclear cells (PBMCs) infected with SARS-CoV-2, as well as on SARS-CoV and MERS-CoV, to uncover shared pathways and elucidate their common disease progression and viral replication mechanisms. We developed CompCorona, the first interactive online tool for visualizing gene response variance among the family Coronaviridae through 2D and 3D principal component analysis (PCA) and exploring systems biology variance using pathway plots. We also made preprocessed datasets of lungs and PBMCs infected by SARS-CoV-2, SARS-CoV, and MERS-CoV publicly available through CompCorona. One remarkable finding from the lung and PBMC datasets for infections by SARS-CoV-2, but not infections by other coronaviruses (CoVs), was the significant downregulation of the angiogenin (ANG) and vascular endothelial growth factor A (VEGFA) genes, both directly involved in epithelial and vascular endothelial cell dysfunction. Suppression of the TNF signaling pathway was also observed in cells infected by SARS-CoV-2, along with simultaneous activation of complement and coagulation cascades and pertussis pathways. The ribosome pathway was found to be universally suppressed across all three viruses. The CompCorona online tool enabled the comparative analysis of 9 preprocessed host transcriptome datasets of cells infected by CoVs, revealing the specific host response differences in cases of SARS-CoV-2 infection. This included identifying markers of epithelial dysfunction via interactive 2D and 3D PCA, Venn diagrams, and pathway plots. Our findings suggest that infection by SARS-CoV-2 might induce pulmonary epithelial dysfunction, a phenomenon not observed in cells infected by other CoVs. The publicly available CompCorona tool, along with the preprocessed datasets of cells infected by various CoVs, constitutes a valuable resource for further research into CoV-associated syndromes.

Population-level integration of single-cell datasets enables multi-scale analysis across samples

The increasing generation of population-level single-cell atlases has the potential to link sample metadata with cellular data. Constructing such references requires integration of heterogeneous cohorts with varying metadata. Here we present single-cell population level integration (scPoli), an open-world learner that incorporates generative models to learn sample and cell representations for data integration, label transfer and reference mapping. We applied scPoli on population-level atlases of lung and peripheral blood mononuclear cells, the latter consisting of 7.8 million cells across 2,375 samples. We demonstrate that scPoli can explain sample-level biological and technical variations using sample embeddings revealing genes associated with batch effects and biological effects. scPoli is further applicable to single-cell sequencing assay for transposase-accessible chromatin and cross-species datasets, offering insights into chromatin accessibility and comparative genomics. We envision scPoli becoming an important tool for population-level single-cell data integration facilitating atlas use but also interpretation by means of multi-scale analyses.

Moderate hypoxia induces metabolic divergence in circulating monocytes and tissue resident macrophages from Berkeley sickle cell anemia mice.

Human and murine sickle cell disease (SCD) associated pulmonary hypertension (PH) is defined by hemolysis, nitric oxide depletion, inflammation, and thrombosis. Further, hemoglobin (Hb), heme, and iron accumulation are consistently observed in pulmonary adventitial macrophages at autopsy and in hypoxia driven rodent models of SCD, which show distribution of ferric and ferrous Hb as well as HO-1 and ferritin heavy chain. The anatomic localization of these macrophages is consistent with areas of significant vascular remodeling. However, their contributions toward progressive disease may include unique, but also common mechanisms, that overlap with idiopathic and other forms of pulmonary hypertension. These processes likely extend to the vasculature of other organs that are consistently impaired in advanced SCD. To date, limited information is available on the metabolism of macrophages or monocytes isolated from lung, spleen, and peripheral blood in humans or murine models of SCD. Here we hypothesize that metabolism of macrophages and monocytes isolated from this triad of tissue differs between Berkley SCD mice exposed for ten weeks to moderate hypobaric hypoxia (simulated 8,000 ft, 15.4% O2) or normoxia (Denver altitude, 5000 ft) with normoxia exposed wild type mice evaluated as controls. This study represents an initial set of data that describes the metabolism in monocytes and macrophages isolated from moderately hypoxic SCD mice peripheral lung, spleen, and blood mononuclear cells.

Aerosol vaccination of chicken pullets with irradiated avian pathogenic Escherichia coli induces a local immunostimulatory effect.

The present study investigated the expression of cytokines and cellular changes in chickens following vaccination with irradiated avian pathogenic Escherichia coli (APEC) and/or challenge. Four groups of 11-week-old pullets, each consisting of 16 birds were kept separately in isolators before they were sham inoculated (N), challenged only (C), vaccinated (V) or vaccinated and challenged (V+C). Vaccination was performed using irradiated APEC applied via aerosol. For challenge, the homologous strain was administered intratracheally. Birds were sacrificed on 3, 7, 14 and 21 days post challenge (dpc) to examine lesions, organ to body weight ratios and bacterial colonization. Lung and spleen were sampled for investigating gene expression of cytokines mediating inflammation by RT-qPCR and changes in the phenotype of subsets of mononuclear cells by flow cytometry. After re-stimulation of immune cells by co-cultivation with the pathogen, APEC-specific IFN-γ producing cells were determined. Challenged only birds showed more severe pathological and histopathological lesions, a higher probability of bacterial re-isolation and higher organ to body weight ratios compared to vaccinated and challenged birds. In the lung, an upregulation of IL-1β and IL-6 following vaccination and/or challenge at 3 dpc was observed, whereas in the spleen IL-1β was elevated. Changes were observed in macrophages and TCR-γδ+ cells within 7 dpc in spleen and lung of challenged birds. Furthermore, an increase of CD4+ cells in spleen and a rise of Bu-1+ cells in lung were present in vaccinated and challenged birds at 3 dpc. APEC re-stimulated lung and spleen mononuclear cells from only challenged pullets showed a significant increase of IFN-γ+CD8α+ and IFN-γ+TCR-γδ+ cells. Vaccinated and challenged chickens responded with a significant increase of IFN-γ+CD8α+ T cells in the lung and IFN-γ+TCR-γδ+ cells in the spleen. Re-stimulation of lung mononuclear cells from vaccinated birds resulted in a significant increase of both IFN-γ+CD8α+ and IFN-γ+TCR-γδ+ cells. In conclusion, vaccination with irradiated APEC caused enhanced pro-inflammatory response as well as the production of APEC-specific IFN-γ-producing γδ and CD8α T cells, which underlines the immunostimulatory effect of the vaccine in the lung. Hence, our study provides insights into the underlying immune mechanisms that account for the defense against APEC.

Resveratrol attenuates staphylococcal enterotoxin B-activated immune cell metabolism via upregulation of miR-100 and suppression of mTOR signaling pathway.

Acute Respiratory Distress Syndrome (ARDS) is triggered by a variety of insults, such as bacterial and viral infections, including SARS-CoV-2, leading to high mortality. In the murine model of ARDS induced by Staphylococcal enterotoxin-B (SEB), our previous studies showed that while SEB triggered 100% mortality, treatment with Resveratrol (RES) completely prevented such mortality by attenuating inflammation in the lungs. In the current study, we investigated the metabolic profile of SEB-activated immune cells in the lungs following treatment with RES. RES-treated mice had higher expression of miR-100 in the lung mononuclear cells (MNCs), which targeted mTOR, leading to its decreased expression. Also, Single-cell RNA-seq (scRNA seq) unveiled the decreased expression of mTOR in a variety of immune cells in the lungs. There was also an increase in glycolytic and mitochondrial respiration in the cells from SEB + VEH group in comparison with SEB + RES group. Together these data suggested that RES alters the metabolic reprogramming of SEB-activated immune cells, through suppression of mTOR activation and its down- and upstream effects on energy metabolism. Also, miR-100 could serve as novel potential therapeutic molecule in the amelioration of ARDS.

In Vitro and In Vivo Relevant Antineoplastic Activity of Platinum(II) Complexes toward Triple-Negative MDA-MB-231 Breast Cancer Cell Line.

Two platinum complexes [Pt(HL3)Cl]·H2O (3) and [Pt(HL4)Cl]·H2O (4) containing α- and β-naphthyl groups, respectively, were investigated in more detail in vitro and in vivo for antineoplastic activity. The cytotoxicity activity induced by these platinum(II) compounds against breast cancer (MDA-MB-231 and MCF-7), lung (A549), prostate (PC3), pancreas (BXPC-3), and normal peripheral blood mononuclear (PBMC) cells were evaluated by MTT assay. The cell viability MTT assay showed that complex (4) was more cytotoxic to all cancer cell lines tested and less cytotoxic against human PBMC. Therefore, complex (4) was selected to further investigate the mechanism of cytotoxic effects involved against MDA-MB-231 cell line (human triple-negative breast cancer). Sub-G1 analysis of the cell cycle showed that this complex induces cell death by apoptosis due to the cell loss of DNA content detected in flow cytometry. The cytotoxic effect induced by complex (4) was associated with the capability of the complex to induce mitochondrial membrane depolarization, as well as increase ROS levels and caspase activation, as a result of the activation of both extrinsic and intrinsic apoptosis pathways. Ultrastructural alterations were observed using scanning and transmission electron microscopy (SEM and TEM), such as membrane blebbing, filopodia reduction, empty mitochondrial matrix, and DNA fragmentation. Furthermore, complex (4) was tested in an MDA-MB-231 tumor nodule xenograft murine model and demonstrated a remarkable reduction in tumor size in BALB/c nude mice, when compared to the control animals.

Production of interferon gamma and interleukin 17A in chicken T-cell subpopulations hallmarks the stimulation with live, irradiated and killed avian pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) causes colibacillosis with different clinical manifestations. The disease is associated with compromised animal welfare and results in substantial economic losses in poultry production worldwide. So far, immunological mechanisms of protection against colibacillosis are not comprehensively resolved. Therefore, the present study aimed to use an ex vivo model applying chicken mononuclear cells stimulated by live and inactivated APEC. For this purpose, an 8-color flow cytometry panel was set up to target viable chicken immune cells including CD45+, CD8α+, CD4+, TCR-γδ+, Bu-1+ cells and monocytes/macrophages along with the cytokines interferon gamma (IFN-γ) or interleukin 17A (IL-17A). The 8-color flow cytometry panel was applied to investigate the effect of live and two different types of inactivated APEC (formalin-killed APEC and irradiated APEC) on the cellular immune response. For that, mononuclear cells from spleen, lung and blood of 10-week-old specific pathogen-free layer birds were isolated and stimulated with live, irradiated or killed APEC. Intracellular cytokine staining and RT-qPCR assays were applied for the detection of IFN-γ and IL-17A protein level, as well as at mRNA level for spleenocytes. Ex vivo stimulation of isolated splenocytes, lung and peripheral blood mononuclear cells (PBMCs) from chickens with live, irradiated or killed APEC showed an increasing number of IFN-γ and IL-17A producing cells at protein and mRNA level. Phenotyping of the cells from blood and organs revealed that IFN-γ and IL-17A were mainly produced by CD8α+, TCR-γδ+ T cells as well as CD4+ T cells following stimulation with APEC. Expression level of cytokines were very similar following stimulation with live and irradiated APEC but lower when killed APEC were applied. Consequently, in the present study, an ex vivo model using mononuclear cells of chickens was applied to investigate the cellular immune response against APEC. The results suggest the relevance of IFN-γ and IL-17A production in different immune cells following APEC infection in chickens which needs to be further investigated in APEC primed birds.

Selection of human single domain antibodies (sdAb) against thymidine kinase 1 and their incorporation into sdAb-Fc antibody constructs for potential use in cancer therapy.

Thymidine Kinase 1 (TK1) is primarily known as a cancer biomarker with good prognostic capabilities for both hematological and solid malignancies. However, recent studies targeting TK1 at protein and mRNA levels have shown that TK1 may be useful as a therapeutic target. In order to examine the use of TK1 as a therapeutic target, it is necessary to develop therapeutics specific for it. Single domain antibodies (sdAbs), represent an exciting approach for the development of immunotherapeutics due to their cost-effective production and higher tumor penetration than conventional antibodies. In this study, we isolated sdAb fragments specific to human TK1 from a human sdAb library. A total of 400 sdAbs were screened through 5 rounds of selection by monoclonal phage ELISA. The most sensitive sdAb fragments were selected as candidates for preclinical testing. The sdAb fragments showed specificity for human TK1 in phage ELISA, Western blot analysis and had an estimated limit of detection of 3.9 ng/ml for the antibody fragments 4-H-TK1_A1 and 4-H-TK1_D1. The antibody fragments were successfully expressed and used for detection of membrane associated TK1 (mTK1) through flow cytometry on cancer cells [lung (~95%), colon (~87%), breast (~53%)] and healthy human mononuclear cells (MNC). The most sensitive antibody fragments, 4-H-TK1_A1 and 4-H-TK1_D1 were fused to an engineered IgG1 Fc fragment. When added to cancer cells expressing mTK1 co-cultured with human MNCs, the anti-TK1-sdAb-IgG1_A1 and D1 were able to elicit a significant antibody-dependent cell-mediated cytotoxicity (ADCC) response against lung cancer cells compared to isotype controls (P<0.0267 and P<0.0265, respectively). To our knowledge this is the first time that the isolation and evaluation of human anti-TK1 single domain antibodies using phage display technology has been reported. The antibody fragments isolated here may represent a valuable resource for the detection and the targeting of TK1 on tumor cells.

Metabolic reprogramming in pulmonary arterial hypertension: is it a cancer-like disease?

Abstract Background Idiopathic pulmonary arterial hypertension (iPAH) is a rare and chronic disease associated with poor outcomes. Previously considered a disease restricted to the pulmonary circulation, PAH is now being recognized as a systemic disorder that is associated with metabolic dysfunction. The aim of this study is to analyze the metabolic reprogramming in the lung and peripheral blood mononuclear cell (PBMCs) of iPAH patients and explore their potential roles in PAH pathophysiology. Methods Five independent datasets, containing transcriptomic data of human PBMCs (GSE22356 and GSE33463) and lung (GSE48149 GSE113439 and GSE117261) samples, from 139 iPAH patients and 96 healthy controls, were downloaded at the GEO database. In each dataset, the samples were normalized and a pair-wise comparison between control and iPAH samples was performed using limma package, for the R programming language. Genes with a p-value lower than 0.05 were considered differentially expressed between the two groups. A subset of metabolism related genes was selected, and their expression was compared across the datasets. Results Among the 13 genes with differential expression identified, only 10 had a coherent expression across all datasets (Figure 1). Firstly, we report an association with insulin resistance through impairment of PI3K signaling in iPAH patients, by expressing lower levels of the heterodimer PIK3CD and regulatory PIK3IP1 and PIKR1 subunits in PBMCs, and by expressing higher levels of its downstream targets in the lung (TBC1D4). However, more extensive metabolic dysfunction was observed. A significant glycolytic shift in the lung and PBMCs was present, as a consequence of deregulation in genes involved in aerobic glycolysis and decreased fatty acid oxidation, namely increased expression of PD1K and lower levels of expression of LDHB. The findings of decreased SLC25A1 protein in both PBMCs and lung suggest impairment of the tricarboxylic acid (TCA) cycle flux in PAH. Additionally, SLC1A5 highlights the involvement of glutamine metabolism and glutaminolysis derangements in PAH. Conversely, SREBP1 is involved in sterol biosynthesis and lower levels in PMBCs results in impaired resolution of inflammatory responses. Finally, although the role of autophagy in iPAH is complex, higher levels of expression of ATG13 in PBMCs and lower levels in the lung confirm autophagy deregulation in iPAH. Interestingly, all the metabolic pathways identified (Figure 2) are hallmarks of the metabolic reprogramming seen in cancer cells, a finding already suggested by the clonal proliferation of pulmonary artery smooth muscle cells described in plexiform lesions. Conclusion Our results provide novel insights into the metabolic regulation in iPAH. Molecularly, these cells exhibit many features common to cancer cells, suggesting the opportunity to exploit therapeutic strategies used in cancer to treat iPAH. Funding Acknowledgement Type of funding sources: None.

Tissue Resources for the Functional Annotation of Animal Genomes

In order to generate an atlas of the functional elements driving genome expression in domestic animals, the Functional Annotation of Animal Genome (FAANG) strategy was to sample many tissues from a few animals of different species, sexes, ages, and production stages. This article presents the collection of tissue samples for four species produced by two pilot projects, at INRAE (National Research Institute for Agriculture, Food and Environment) and the University of California, Davis. There were three mammals (cattle, goat, and pig) and one bird (chicken). It describes the metadata characterizing these reference sets (1) for animals with origin and selection history, physiological status, and environmental conditions; (2) for samples with collection site and tissue/cell processing; (3) for quality control; and (4) for storage and further distribution. Three sets are identified: set 1 comprises tissues for which collection can be standardized and for which representative aliquots can be easily distributed (liver, spleen, lung, heart, fat depot, skin, muscle, and peripheral blood mononuclear cells); set 2 comprises tissues requiring special protocols because of their cellular heterogeneity (brain, digestive tract, secretory organs, gonads and gametes, reproductive tract, immune tissues, cartilage); set 3 comprises specific cell preparations (immune cells, tracheal epithelial cells). Dedicated sampling protocols were established and uploaded in https://data.faang.org/protocol/samples. Specificities between mammals and chicken are described when relevant. A total of 73 different tissues or tissue sections were collected, and 21 are common to the four species. Having a common set of tissues will facilitate the transfer of knowledge within and between species and will contribute to decrease animal experimentation. Combining data on the same samples will facilitate data integration. Quality control was performed on some tissues with RNA extraction and RNA quality control. More than 5,000 samples have been stored with unique identifiers, and more than 4,000 were uploaded onto the Biosamples database, provided that standard ontologies were available to describe the sample. Many tissues have already been used to implement FAANG assays, with published results. All samples are available without restriction for further assays. The requesting procedure is described. Members of FAANG are encouraged to apply a range of molecular assays to characterize the functional status of collected samples and share their results, in line with the FAIR (Findable, Accessible, Interoperable, and Reusable) data principles.

Beneficial effect of long-chain n-3 polyunsaturated fatty acid supplementation on tuberculosis in mice

Intakes of the omega-3 essential fatty acids (n-3 EFAs) are low in the general adult population, with high n-6/n-3 polyunsaturated fatty acid (PUFA) ratios and the accompanying suboptimal n-3 PUFA status. Eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) have antibacterial and inflammation-resolving effects in tuberculosis (TB). However, whether switching to a diet with optimum n-3 EFA intake after the infection has comparable benefits has not been investigated. We aimed to compare the effects of a diet with sufficient n-3 EFA content in an acceptable n-6/n-3 PUFA ratio for rodents ((n-3)eFAS group) with those on the same diet supplemented with EPA and DHA (EPA/DHA group) in Mycobacterium tuberculosis (Mtb)-infected C3HeB/FeJ mice with a low n-3 PUFA status. Mice were conditioned on an n-3 PUFA-deficient diet with a high n-6/n-3 PUFA ratio for 6 weeks before Mtb infection and randomized to either (n-3)eFAS or EPA/DHA diets 1 week post-infection for 3 weeks. At endpoint, EPA and DHA compositions were higher and arachidonic acid, osbond acid, and total n-6 LCPUFAs lower in all lipid pools measured in the EPA/DHA group (all P < 0.001). Percentage body weight gain was higher (P = 0.017) and lung bacterial load lower (P < 0.001) in the EPA/DHA group. Additionally, the EPA/DHA group had a more pro-resolving lung lipid mediator profile and lower lung in IL-1α and IL-1β concentrations (P = 0.023, P = 0.049). Inverse correlations were found between the lung and peripheral blood mononuclear cell EPA and DHA and selected pro-inflammatory cytokines. These are the first findings that indicate that EPA/DHA supplementation provides benefits superior to a diet with sufficient n-3 EFAs concerning bacterial killing, weight gain and lung inflammation resolution in Mtb-infected mice with a low n-3 PUFA status. Therefore, EPA and DHA may be worth considering as adjunct TB treatment.

A single-cell atlas of the lung mononuclear cell response in Staphylococcus Enterotoxin B-induced Acute Respiratory Distress Syndrome

Abstract The emergence of SARS-CoV-2 variants will change the landscape of what we already know about COVID-19, making it important to understand the immunological and molecular mechanism of this illness. The virulent nature of the virus, disease outcomes and lack of suitable facilities for studying virus-infected samples has made the need for small animal models urgent. Staphylococcus enterotoxin B (SEB)-induced Acute Respiratory Distress Syndrome (ARDS) mimics the cytokine storm presented in patients with severe COVID-19 and is a safer alternative than working with infected samples. To identify pathways in pulmonary cells that potentially cause ARDS, we utilized single-cell RNA sequencing (scRNASeq) to profile the whole lung cells of C3H/HeJ mice treated with SEB and healthy controls. While it is well established that SEB induces robust proliferation of CD4+ T cells with the receptor Vβ8.2 by binding of these cells with antigen presenting cells, effects in other cell types is not well described. We found that intranasal administration of SEB induced the proliferation of three subsets of macrophages, neutrophils, and cytotoxic T cells in the lungs. Our data also illustrated a loss of multiple cell types including resident macrophages, multiple subsets of B cells, and club cells. Macrophage subsets displayed increased expression of genes related to interferon signaling and lipid metabolism. This data emphasizes the importance of understanding the role of macrophages in ARDS, and can further provide insights into elucidating the mechanisms not only in SEB-induced ARDS but also in severe COVID-19. This may serve as a model for severe case of the disease. (Supported by NIH P01AT003961, P20GM103641, R01AI129788, R01 ES030144 and R01AI123947)

Butyrate mediates the amelioration of Staphylococcal Enterotoxin B-induced Acute Respiratory Distress Syndrome by downregulating the expression of Histone Deacetylases

Abstract Despite rigorous global containment and quarantine efforts that have been applied globally to control SARS-CoV-2, the spreading of this contagious disease has accelerated all over the world. In our study, we used staphylococcal enterotoxin B (SEB), which is a superantigen can lead to toxic shock syndrome in C57BL/6 murine model and thus, mimics COVID19. In the current study, we investigated the suppressive effect of sodium butyrate (BUT) on a murine model of SEB-induced Acute Respiratory Distress Syndrome (ARDS). The data shows that supplementation with BUT at a dose of 200 mg/kg orally reduced overall ARDS severity. Plethysmography analysis data indicated that sodium butyrate improves the clinical functions of the lungs. In addition, our flow cytometry data indicated that BUT mediated the suppression of lung mononuclear cells such as CD4+ T cells, CD8+ T cells, VB8+ T cells and NK-T cells while at the same time increasing T regulatory cells. Cytokine storm is one of the most important challenge in COVID19 cases, in our study, we have found that the proinflammatory cytokines such as IL-2, TNFα and IFNγ in broncho-alveolar lavage fluid (BALF) significantly decreased in SEB+BUT compared to SEB+VEH as detected by ELISA. Additionally, histopathological analysis of H &amp; E stained lungs indicated that BUT decreased the level of infiltration of inflammatory cells in the lungs of SEB+BUT compared to SEB+VEH mice. Additionally, we investigated the role of BUT on the expression of histone deacetylases (HDACs). Collectively, our data suggested that BUT ameliorated SEB-induced ARDS by downregulating the expression of class I and Class II HDACs which mediate the increase in T regulatory cells that suppress the inflammatory response in SEB-induced ARDS.

Calves Infected with Virulent and Attenuated Mycoplasma bovis Strains Have Upregulated Th17 Inflammatory and Th1 Protective Responses, Respectively.

Mycoplasma bovis is a critical bovine pathogen, but its pathogenesis remains poorly understood. Here, the virulent HB0801 (P1) and attenuated HB0801-P150 (P150) strains of M. bovis were used to explore the potential pathogenesis and effect of induced immunity from calves’ differential transcriptomes post infection. Nine one-month-old male calves were infected with P1, P150, or mock-infected with medium and euthanized at 60 days post-infection. Calves in P1 group exhibited other clinical signs and pathological changes compared to the other two groups. Transcriptome profiles of peripheral blood mononuclear cells revealed seven and 10 hub differentially expressed genes (DEGs) in P1 and P150 groups compared with mock-infected group, respectively. Then, P1-induced pathogenesis was predicted to be associated with enhanced Th17, and P150-induced immunity with Th1 response and expression of ubiquitination-associated enzymes. Association analysis showed that 14 and 11 DEGs were positively and negatively correlated with pathological changes, respectively. Furthermore, up-regulated expression in molecules critical to differentiation of pathogenic Th17 cells in lung and peripheral blood mononuclear cells in P1 group was validated at RNA and protein levels. The results confirmed virulent and attenuated strains might be associated with biased differentiation of pro-inflammatory pathogenic Th17 and Th1 subsets respectively.

Heterogeneous expression of Toll-like receptors genes in lymphoid tissues of different ages pigs

Toll-like receptors (TLRs), known as the pathogen recognition receptors, recognize the microbial components. The details expression kinetics of TLRs may help to understand the immune responsiveness of pigs, but the expression patterns of all TLRs (TLR1–10) have not yet been studied in pigs. Therefore, this study unravels the expression patterns of the TLR family set of genes (TLR1–10) in different lymphoid tissues collected from pigs at different ages. A total of nine clinically healthy Pietrain pigs at three age groups were selected for this experiment. Each age group consisted of three animals. Cervical lymph node (CLN), thymus, liver, spleen, lung, heart, skin tissue and peripheral blood mononuclear cells (PBMC) were collected for both mRNA and protein isolation. The mRNA expression of TLRs (1−10) was quantified in all tissues. TLR3 mRNA was the most abundant in all tissues. It was expressed highly in thymus, kidney, lungs and liver tissues. Western blot and immunofluorescence was performed for protein expression and localization of selected TLRs (TLR2, 3 and 9) in selected tissues (CLN, spleen and lungs). The Western blot results of TLR2, 3 and 9 in selected tissues appeared to be consistent with the mRNA expression results. Cells in lungs, spleen and CLN were positively immunostained for TLR2, 3 and 9. This study sheds light on the expression patterns of TLR (1–10) genes in important lymphoid tissues in pigs of different ages. The heterogeneous expression of TLRs with ages may contribute to the altered immune responsiveness of pigs with ages.

Preventative tracheal administration of interleukin-27 attenuates allergic asthma by improving the lung Th1 microenvironment.

Interleukin-27 (IL-27) modulates CD4+ T-cell differentiation and function. The aim of this study is to investigate the effect and molecular mechanisms of IL-27 on the development of asthma. IL-27 was intranasally administered in an ovalbumin-induced asthma model, and lung mononuclear cells and different Th cell classes were detected by fluorescence-activated cell sorting. The effect and mechanisms of IL-27 on human bronchial epithelial (HBE) cells were investigated by measuring changes in chemotactic factors, cytokines, transcription factors, and signaling pathways. We found that intranasal administration of IL-27 could attenuate airway inflammation and hyperresponsiveness, upregulate the type 1 T helper(Th1)-T memory (Tm) cells and regulatory T (Treg) cells subgroups of lung tissue lymphocytes, and diminish the levels of type 2 T helper (Th2) cytokines. IL-27 upregulated the expression of C-C motif chemokine ligand 2 (CCL2), CCL3, and CCL4 in HBE cells and promoted the production of chemotactic factors to attract monocyte recruitment. Recruited monocytes secondarily secreted IL-27 to influence HBE cells in a positive feedback cycle. After IL-27 intervention, signal transducer and activator of transcription 1 (STAT1) phosphorylation increased, while STAT4 and STAT6 phosphorylation declined. Preventative intranasal administration of IL-27 can recruit more IL-27-secreted monocytes to the airway and change the different T-cell classes in lung. The improved Th1 environment helps to alleviate Th2-mediated allergic asthma by repairing the STAT1 pathway but not the STAT4 pathway.

Evolutional conservation of molecular structure and antiviral function of a type I interferon, IFN-kappa, in poultry

IFN-kappa (IFN-κ) is a type I IFN expressed by keratinocytes, monocytes and dendritic cells with important roles during the innate immune response period. This research was conducted to elaborate the evolution and characteristics of IFN-κ in poultry. Chicken IFN-κ is located on the sex-determining Z chromosome, which is greatly different from mammals. Poultry IFN-κ cluster together in a species-specific manner through positive selection pressure and share only 19–33% homology with mammalian IFN-κ and poultry other type I IFN. Both chicken and duck IFN-κ was constitutively expressed in spleen, skin, lung, and peripheral blood mononuclear cells (PBMC), as well as being significantly induced after treatment with virus in PBMC. Biologically, poultry IFN-κ has antiviral activity against VSV in chicken embryonic fibroblasts and duck embryonic fibroblasts (CEF and DEF) cells, and induces the expression of IFN stimulated genes (ISGs). After treatment with JAK1 inhibitor, the ISGs expression can be down-regulated. Overall, our research on poultry IFN-κ not only enriches the knowledge about IFN-κ but also facilitates further research on the role of type I IFNs in antiviral defense responses in poultry.

Molecular cloning and functional analysis of peafowl (Pavo cristatus) Toll-like receptor 7.

In order to clone the peafowl (Pavo cristatus) Toll-like receptor 7 (TLR7) gene and study its biological function, the peafowl TLR7 coding sequences (CDS) were amplified by PCR of cDNA from the whole spleen of peafowl. The full-length sequence of the peafowl TLR7 gene CDS is 3,141bp and encodes a 1,046-amino acid protein with a classic TLR composition of 16 leucine-rich repeats (LRR). Insertions of amino acids were found at position 15 of LRR2, LRR5, LRR7, LRR9, LRR11, LRR12, LRR14, and LRR15; and position 10 of LRR11. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that the peafowl TLR7 gene was highly expressed in lymphoid tissues of the spleen, bursa, bone marrow, lung, and peripheral blood mononuclear cells (PBMC). HEK293T cells were transfected with a peafowl TLR7 plasmid, and functional analysis showed that peafowl TLR7 could respond to R848, leading to activation of NF-κB. Following R848 stimulation or Newcastle disease virus infection of peafowl PBMC, the levels of IL-1β, IFN-γ, CCLi2, and TGF-β4 mRNA, assessed by quantitative real-time PCR, increased significantly. Triggering peafowl TLR7 results in upregulation of inflammatory cytokines and chemokines, suggesting that peafowl TLR7 plays an important role in the innate immune response.

Prenatal tobacco smoke exposure predisposes offspring mice to exacerbated allergic airway inflammation associated with altered innate effector function

BackgroundEpidemiological studies suggest that prenatal and early life environmental exposures have adverse effects on pulmonary function and are important contributors in the development of childhood asthma and allergic disease. The mechanism by which environmental tobacco smoke (ETS) exposure in utero promotes the development of allergic asthma remains unclear. In this study, we investigated the immunological consequences of prenatal exposure to ETS in order to understand events responsible for the development or exacerbation of allergic asthma.MethodsPregnant C57BL/6 mice were exposed to either ETS or filtered air throughout gestation and the effect on pulmonary inflammation in the offspring were examined and compared. Specifically, the effects on eosinophilic inflammation, airway hyperreactivity, goblet cell hyperplasia, properties of pulmonary natural killer (NK) cells and type 2 cytokines elicited in response to inhaled house dust mite (HDM) allergen were investigated in the progeny.ResultsExposure to ETS prenatally significantly exacerbated HDM-induced airway eosinophilic inflammation, hyperreactivity, mucus secretion, cysteinyl leukotriene biosynthesis and type 2 cytokine production in the offspring. Consistently, lung mononuclear cells from ETS-exposed offspring secreted higher levels of IL-13 when stimulated in vitro with anti-αβ TCR antibody or HDM allergen. Moreover, offspring from ETS-exposed dams exhibited a higher frequency of CD11b+ dendritic cells and CD3+CD4+ T lymphocytes in the lungs following allergen inhalation compared to air-exposed mice. Unexpectedly, the exacerbated allergic inflammation in the ETS-exposed offspring was associated with a reduction in CD3−CD19−NK1.1+CD94+ NK cell numbers and their IFN-γ production, highlighting a role for altered innate immunity in the enhanced allergic response.ConclusionOur results reveal that prenatal exposure to ETS predisposes offspring to an exacerbated allergic airway inflammation that is associated with a reduction in pulmonary NK cell function, suggesting that NK cells play a key role in controlling asthma severity.

Overexpression of IL-38 protein in anticancer drug-induced lung injury and acute exacerbation of idiopathic pulmonary fibrosis.

Interleukin (IL)-38, a member of the IL-1 family, shows high homology to IL-1 receptor antagonist (IL-1Ra) and IL-36 receptor antagonist (IL-36Ra). Its function in interstitial lung disease (ILD) is still unknown. To determine the expression pattern of IL-38 mRNA, a panel of cDNAs derived from various tissues was analyzed by quantitative real-time PCR. Immunohistochemical reactivity with anti-human IL-38 monoclonal antibody (clone H127C) was evaluated semi-quantitatively in lung tissue samples from 12 patients with idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP), 5 with acute exacerbation of IPF, and 10 with anticancer drug-induced ILD (bleomycin in 5 and epidermal growth factor receptor-tyrosine kinase inhibitor in 5). Control lung tissues were obtained from areas of normal lung in 22 lung cancer patients who underwent extirpation surgery. IL-38 transcripts were strongly expressed in the lung, spleen, synoviocytes, and peripheral blood mononuclear cells, and at a lower level in pancreas and muscle. IL-38 protein was not strongly expressed in normal pulmonary alveolar tissues in all 22 control lungs. In contrast, IL-38 was overexpressed in the lungs of 4 of 5 (80%) patients with acute IPF exacerbation and 100% (10/10) of the patients with drug-induced ILD. IL-38 overexpression was limited to hyperplastic type II pneumocytes, which are considered to reflect regenerative change following diffuse alveolar damage in ILD. IL-38 may play an important role in acute and/or chronic inflammation in anticancer drug-induced lung injury and acute exacerbation of IPF.