Activated CD4 T cells connect to airway smooth muscle cells (ASMCs) in vitro via lymphocyte-derived membrane conduits (LMCs) structurally similar to membrane nanotubes with unknown intercellular ...signals triggering their formation. We examined the structure and function of CD4 T cell-derived LMCs, and we established a role for ASMC-derived basic fibroblast growth factor 2 (FGF2b) and FGF receptor (FGFR)1 in LMC formation. Blocking FGF2b's synthesis and FGFR1 function reduced LMC formation. Mitochondrial flux from ASMCs to T cells was partially FGF2b and FGFR1 dependent. LMC formation by CD4 T cells and mitochondrial transfer from ASMCs was increased in the presence of asthmatic ASMCs that expressed more mRNA for FGF2b compared with normal ASMCs. These observations identify ASMC-derived FGF2b as a factor needed for LMC formation by CD4 T cells, affecting intercellular communication.
Varying concentrations of lipopolysaccharide (LPS) in ovalbumin (OVA) may influence the airway response to allergic sensitization and challenge. We assessed the contribution of LPS to allergic airway ...inflammatory responses following challenge with LPS-rich and LPS-free commercial OVA. BALB/c mice were sensitized with LPS-rich OVA and alum and then underwent challenge with the same OVA (10 µg intranasally) or an LPS-free OVA. Following challenge, bronchoalveolar lavage (BAL), airway responsiveness to methacholine and the lung regulatory T cell population (Treg) were assessed. Both OVA preparations induced BAL eosinophilia but LPS-rich OVA also evoked BAL neutrophilia. LPS-free OVA increased interleukin (IL)-2, IL-4 and IL-5 whereas LPS-rich OVA additionally increased IL-1β, IL-12, IFN-γ, TNF-α and KC. Both OVA-challenged groups developed airway hyperresponsiveness. TLR4-deficient mice challenged with either OVA preparation showed eosinophilia but not neutrophilia and had increased IL-5. Only LPS-rich OVA challenged mice had increased lung Tregs and LPS-rich OVA also induced in vitro Treg differentiation. LPS-rich OVA also induced a Th1 cytokine response in human peripheral blood mononuclear cells.We conclude that LPS-rich OVA evokes mixed Th1, Th2 and innate immune responses through the TLR-4 pathway, whereas LPS-free OVA evokes only a Th2 response. Contaminating LPS is not required for induction of airway hyperresponsiveness but amplifies the Th2 inflammatory response and is a critical mediator of the neutrophil, Th1 and T regulatory cell responses to OVA.
Exposure of mice to high concentrations of chlorine leads to the synthesis of cysteinyl leukotrienes (cysLTs). CysLTs contribute to chlorine-induced airway hyperresponsiveness. The aim of the current ...study was to determine the cellular source of the cysLTs. To achieve this aim, we exposed mice to 100 ppm of chlorine for 5 minutes. Intranasal instillation of clodronate in liposomes and of diphtheria toxin in CD11c-DTR mice was used to deplete macrophages. CCR2
mice were used to assess the contribution of recruited macrophages. Eosinophils and neutrophils were depleted with specific antibodies. Platelet-neutrophil aggregation was prevented with an antibody against P-selectin. The potential roles of phagocytosis of neutrophils by macrophages and of transcellular metabolism between epithelial cells and neutrophils were explored in coculture systems. We found that depletion of neutrophils was the only intervention that inhibited the synthesis of cysLTs at 24 hours after chlorine exposure. Although macrophages did synthesize cysLTs in response to phagocytosis of neutrophils, depletion of macrophages did not reduce the increment in cysLTs triggered by chlorine exposure. However, coculture of airway epithelial cells with neutrophils resulted in a significant increase in the synthesis of cysLTs, dependent on the expression of 5-lipoxygenase by neutrophils. We conclude that cysLT synthesis following chlorine exposure may be dependent on transcellular metabolism by neutrophil-epithelial interactions.
Nucleic Acid Sensing in Allergic Disorders Farahnak, Soroor; Chronopoulos, Julia; Martin, James G
International review of cell and molecular biology,
2019, Volume:
345
Journal Article
Peer reviewed
Recent advances indicate that there is crosstalk between allergic disorders and nucleic acid sensing. Triggers that activate inflammatory mechanisms via nucleic acid sensors affect both allergic ...phenotypes and anti-viral responses, depending on the timing and the order of exposure. Viral respiratory infections, such as those caused by the rhinovirus, influenza, and respiratory syncytial virus, are the most frequent cause of significant asthma exacerbations through effects mediated predominantly by TLR3. However, agonists of other nucleic acid sensors, such as TLR7/8 and TLR9 agonists, may inhibit allergic inflammation and reduce clinical manifestations of disease. The allergic state can predispose the immune system to both exaggerated responses to viral infections or protection from anti-viral inflammatory responses. T
2 cytokines appear to alter the epithelium, leading to defective viral clearance or exaggerated responses to viral infections. However, a T
2 skewed allergic response may be protective against a T
1-dependent inflammatory anti-viral response. This review briefly introduces the receptors involved in nucleic acid sensing, addresses mechanisms by which nucleic acid sensing and allergic responses can counteract one another, and discusses the strategies in experimental settings, both in animal and human studies, to harness the nucleic acid sensing machinery for the intervention of allergic disorders.
CD4 T cells express the epidermal growth factor (EGF) receptor ligand, heparin-binding EGF (HB-EGF), with no defined immuno-pathophysiological function. Therefore, we wished to elucidate the function ...of HB-EGF synthesized by CD4 T cells in the context of allergic pulmonary inflammation and the asthma surrogate, airway hyperresponsiveness, in a murine acute model of asthma. In this study, we show how knocking out HB-EGF expression in CD4 T cells in vivo attenuates IL-5 synthesis in the lung that is accompanied by diminished eosinophilic inflammation and airway hyperresponsiveness. HB-EGF coimmunoprecipitates with the transcriptional repressor B cell lymphoma 6 (Bcl-6) in CD4 T cells. Knocking out HB-EGF in CD4 T cells resulted in increased Bcl-6 binding to the IL-5 gene and decreased IL-5 mRNA expression. Thus, these findings suggest an immunoregulatory function for intrinsic HB-EGF expressed by CD4 T cells in T
2 inflammation and airway dysfunction by modulating IL-5 expression via binding to and inhibiting the repressive function of Bcl-6.
Inhalation of organic dust (OD) from swine confinement facilities leads to pulmonary inflammation, airway hyperresponsiveness, and oxidative stress. In mice, pretreatment with a hydroxyl radical ...scavenger prevents airway inflammation and airway hyperresponsiveness (AHR) induced by OD exposure. We sought to determine a mechanism by which OD could induce oxidative stress in bronchial epithelial cells. Human bronchial epithelial cells (BEAS-2B or NHBE) were treated with various concentrations of OD, followed by evaluation of intracellular oxidative stress using 2',7'-dichlorofluorescein diacetate (DCFDA). After stimulation with OD, gene expression of antioxidant genes was assessed by real-time quantitative PCR followed by quantification of Nrf2 nuclear translocation using a luciferase reporter assay. Phagocytic markers (CD36 and CD68) were analyzed by FACS. Cells were treated with an actin inhibitor, cytochalasin D, before OD exposure and evaluated for Nrf2 nuclear translocation and DCFDA. Mice were pretreated with sulforaphane, the Nrf2 activator, before OD exposure and evaluated for pulmonary inflammation and airway reactivity. OD induced a time- and concentration-dependent increase in DCFDA. mRNA expression levels of Nrf2-dependent genes and Nrf2 nuclear translocation were increased after OD exposure. OD exposure increased the expression of CD68 and CD36. Cytochalasin D prevented oxidative stress and Nrf2 nuclear translocation after OD. Pretreatment with sulforaphane prevented OD-induced inflammation and AHR while increasing the uptake of OD in bronchial epithelial cells. Bronchial epithelial cells can phagocytose OD, resulting in an increase in endogenous oxidative stress. Nrf2-dependent mechanisms mediate the antioxidant response to OD.
Clinical studies and experimental animal models conclusively indicate a pivotal role for CD4 T cells in the initiation and exacerbation of allergic asthma. Despite established well-defined functions ...for their various mediators, dictating immunological and structural alterations, there are emerging regulatory mechanisms by which CD4 T cells can potentially exert their pathophysiological properties. Although the expression of heparin binding epidermal growth factor (HB-EGF) by CD4 T cells was initially shown in 1994, there have been no investigations to explore the physiological significance of its synthesis. Here, we studied the role of HB-EGF synthesized by CD4 T cells in a murine model of experimental acute allergic asthma, induced by Ovalbumin, hypothesizing that this growth factor might facilitate the allergic response in the lung, based on the fact that its upregulated expression was observed upon CD4 T cell activation. We found that membrane-bound HB-EGF on CD4 T cells mediates interaction with dendritic cells, which subsequently affects the immune response to allergen. We also found that CD4 derived HB-EGF at the time of allergen challenge augments features of allergic pulmonary disease by mediating eosinophilic inflammation and bronchoconstriction. Therefore, the immunoregulatory role of HB-EGF in allergic asthma provides more insight into the mechanisms by which TH2 driven airway diseases are regulated.CD4 T cells interact with immune and structural cells by direct contact. Previous data have shown that rodent CD4 T cells trigger airway smooth muscle proliferation by mechanisms that involve HB-EGF. One novel mechanism of communication employed by CD4 T cells is the generation of membrane nanotube (MNT)-like structures. Herein, we have demonstrated a molecular mechanism for formation of MNT-like structures through the synthesis of basic fibroblast growth factor 2 (FGF2b) by airway smooth muscle acting on the FGFR1 expressed on the CD4 T cells. The tubular structures mediated mitochondrial transfer from smooth muscle cells to CD4 T cells. The significance of this finding for T cell function is not established. Similarly, the consequences of the interaction for airway smooth muscle function requires further exploration but may contribute to aberrant function of airway smooth muscle in asthma.
Chlorine gas (Cl2) inhalation causes oxidative stress, airway epithelial damage, airway hyperresponsiveness (AHR), and neutrophilia. We evaluated the effect of neutrophil depletion on Cl2-induced AHR ...and its effect on the endogenous antioxidant response, and if eosinophils or macrophages influence Cl2-induced AHR. We exposed male Balb/C mice to 100 ppm Cl2 for 5 minutes. We quantified inflammatory cell populations in bronchoalveolar lavage (BAL), the antioxidant response in lung tissue by quantitative PCR, and nuclear factor (erythroid-derived 2)-like 2 (NRF2) nuclear translocation by immunofluorescence. In vitro, NRF2 nuclear translocation in response to exogenous hypochlorite was assessed using a luciferase assay. Anti-granulocyte receptor-1 antibody or anti-Ly6G was used to deplete neutrophils. The effects of neutrophil depletion on IL-13 and IL-17 were measured by ELISA. Eosinophils and macrophages were depleted using TRFK5 or clodronate-loaded liposomes, respectively. AHR was evaluated with the constant-phase model in response to inhaled aerosolized methacholine. Our results show that Cl2 exposure induced neutrophilia and increased expression of NRF2 mRNA, superoxide dismutase-1, and heme-oxygenase 1. Neutrophil depletion abolished Cl2-induced AHR in large conducting airways and prevented increases in antioxidant gene expression and NRF2 nuclear translocation. Exogenous hypochlorite administration resulted in increased NRF2 nuclear translocation in vitro. After Cl2 exposure, neutrophils occupied 22 ± 7% of the luminal space in large airways. IL-17 in BAL was increased after Cl2, although this effect was not prevented by neutrophil depletion. Neither depletion of eosinophils nor macrophages prevented Cl2-induced AHR. Our data suggest the ability of neutrophils to promote Cl2-induced AHR is dependent on increases in oxidative stress and occupation of luminal space in large airways.
Contact between airway smooth muscle (ASM) cells and activated CD4(+) T cells, a key interaction in diseases such as asthma, triggers ASM cell proliferation and enhances T cell survival. We ...hypothesized that direct contact between ASM and CD4(+) T cells facilitated the transfer of anti-apoptotic proteins via nanotubes, resulting in increased survival of activated CD4(+) T cells. CD4(+) T cells, isolated from PBMCs of healthy subjects, when activated and cocultured with ASM cells for 24 h, formed nanotubes that were visualized by immunofluorescence and atomic force microscopy. Cell-to-cell transfer of the fluorescent dye calcein-AM confirmed cytoplasmic communication via nanotubes. Immunoreactive B cell lymphoma 2 (Bcl-2) and induced myeloid leukemia cell differentiation protein (Mcl-1), two major anti-apoptotic proteins, were present within the nanotubes. Downregulation of Mcl-1 by small interfering RNA in ASM cells significantly increased T cell apoptosis, whereas downregulation of Bcl-2 had no effect. Transfer of GFP-tagged Mcl-1 from ASM cells to CD4(+) T cells via the nanotubes confirmed directionality of transfer. In conclusion, activated T cells communicate with ASM cells via nanotube formation. Direct transfer of Mcl-1 from ASM to CD(+) T cells via nanotubes is involved in T cell survival. This study provides a novel mechanism of survival of CD4(+) T cells that is dependent on interaction with a structural cell.
Sulfonamides exhibit their antitumor effects through multiple mechanisms including inhibition of membrane bound carbonic anhydrases, prevention of microtubule assembly, cell cycle arrest, and ...inhibition of angiogenesis. Here, sulfabenzamide's mechanisms of action on T-47D breast cancer cells were determined. Cells incubated with sulfabenzamide exhibited negligible levels of apoptosis, necrosis and cell cycle arrest when compared to untreated cells. These results were confirmed by morphological examinations, DNA fragmentation assays, flow cytometric and real time RT-PCR analysis. Surprisingly, despite negligible detection of DNA fragmentation, a considerable increase in caspase-3 activity was observed in cells incubated with sulfabenzamide. The increased expression ratio of DFF-45/DFF-40 indicated that caspase-3-related DNA fragmentation was blocked and apoptosis symptoms could not be seen. However, the effects of caspase-3 for PARP1 and DNA-PK deactivation resulted in autophagy induction. The overexpression of critical genes involved in autophagy, including ATG5, p53 and DRAM, indicated that in T-47D cells sulfabenzamide-induced antiproliferative effect was mainly exerted through induction of autophagy. Furthermore, downregulation of AKT1 and AKT2 as well as over expression of PTEN resulted in attenuation of AKT/mTOR survival pathway showing that death autophagy should be occurred in sulfabenzamide treatment.
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