C5a drives airway constriction and inflammation during the effector phase of allergic asthma, mainly through the activation of C5a receptor 1 (C5aR1). Yet, C5aR1 expression on myeloid and lymphoid cells during the allergic effector phase is ill-defined. Recently, we generated and characterized a floxed green fluorescent protein (GFP)-C5aR1 knock-in mouse. Here, we used this reporter strain to monitor C5aR1 expression in airway, pulmonary and lymph node cells during the effector phase of OVA-driven allergic asthma. C5aR1 reporter and wildtype mice developed a similar allergic phenotype with comparable airway resistance, mucus production, eosinophilic/neutrophilic airway inflammation and Th2/Th17 cytokine production. During the allergic effector phase, C5aR1 expression increased in lung tissue eosinophils but decreased in airway and pulmonary macrophages as well as in pulmonary CD11b+ conventional dendritic cells (cDCs) and monocyte-derived DCs (moDCs). Surprisingly, expression in neutrophils was not affected. Of note, moDCs but not CD11b+ cDCs from mediastinal lymph nodes (mLN) expressed less C5aR1 than DCs residing in the lung after OVA challenge. Finally, neither CD103+ cDCs nor cells of the lymphoid lineage such as Th2 or Th17-differentiated CD4+ T cells, B cells or type 2 innate lymphoid cells (ILC2) expressed C5aR1 under allergic conditions. Our findings demonstrate a complex regulation pattern of C5aR1 in the airways, lung tissue and mLN of mice, suggesting that the C5a/C5aR1 axis controls airway constriction and inflammation through activation of myeloid cells in all three compartments in an experimental model of allergic asthma.
Motile cilia perform a range of important mechanosensory and chemosensory functions, along with expulsion of mucus and inhaled pathogens from the lungs. Here we demonstrate that spectral domain optical coherence phase microscopy (SD-OCPM), which combines the principles of optical coherence tomography (OCT) and confocal microscopy, is particularly well-suited for characterization of both morphology and the ciliary dynamics of mouse trachea. We present micro-anatomical images of mouse trachea, where different cell types can be clearly visualized. The phase contrast, which measures the sub-nanometer changes in axial optical pathlength is used to determine the frequency and direction of cilia beatings.
Cell lines and primary cells exhibit varying degrees of resistance to DNA transfection strategies. In this study, we employed the synthetic peptide Tat-RGD (TR), composed of the HIV-1 derived translocation peptide Tat fused to the integrin binding RGD motif, as a tool for improving DNA transfer into pulmonary cells. Binding experiments between DNA and TR and cytotoxicity measurements of TR treated cells were undertaken to optimize DNA and TR concentrations for transfection. Addition of a complex of TR and DNA (TRD) to A549 cells yielded significant transgene expression. When TRD was combined with Lipofectamine (TRDL), the expression was increased by 5-fold over Lipofectamine (DL) and by approximately 30-fold over TRD-mediated transfections. Also, in primary smooth muscle cells (SMC) and fibroblasts (FB) derived from pulmonary arteries, an increase in TRDL-mediated transfection efficiency was observed by a factor of approximately 2 and approximately 3 over that of DL. Laser scanning confocal microscopy for visualizing TR-dependent DNA uptake demonstrated that the internalization of TRDL complexes is linked to caveoli in the plasma membrane. Interfering with caveoli formation by methyl-b-cyclo-dextrin drastically decreased the transfection efficiency by TR. In conclusion, the Tat-RGD peptide mediates efficient gene delivery in human pulmonary cells, in particular when combined with a standard cationic lipid based transfection reagent. The enhancement of DNA uptake by Tat-RGD is suggested to be mediated by caveoli-dependent endocytosis.
We present a forward-viewing fiber scanning endoscope (FSE) for high-speed volumetric optical coherence tomography (OCT). The reduction in size of the probe was achieved by substituting the focusing optics by an all-fiber-based imaging system which consists of a combination of scanning single-mode fibers, a glass spacer, made from a step-index multi-mode fiber, and a gradient-index fiber. A lateral resolution of 11 μm was achieved at a working distance of 1.2 mm. The newly designed piezo-based FSE has an outer diameter of 1.6 mm and a rigid length of 13.5 mm. By moving the whole imaging optic in spirals for scanning the sample, the beam quality remains constant over the entire field of view with a diameter of 0.8 mm. The scanning frequency was adjusted to 1.22 kHz for use with a 3.28 MHz Fourier domain mode locked OCT system. Densely sampled volumes have been imaged at a rate of 6 volumes per second.
Pulmonary eosinophils comprise at least two distinct populations of resident eosinophils (rEOS) and inflammatory eosinophils (iEOS), the latter recruited in response to pulmonary inflammation. Here, we determined the impact of complement activation on rEOS and iEOS trafficking and function in two models of pulmonary inflammation.BALB/c wild-type and C5ar1-/- mice were exposed to different allergens or IL-33. Eosinophil populations in the airways, lung, or mediastinal lymph nodes (mLN) were characterized by FACS or immunohistochemistry. rEOS and iEOS functions were determined in vivo and in vitro.HDM and IL-33 exposure induced a strong accumulation of iEOS but not rEOS in the airways, lungs, and mLNs. rEOS and iEOS expressed C3/C5 and C5aR1, which were significantly higher in iEOS. Initial pulmonary trafficking of iEOS was markedly reduced in C5ar1-/- mice and associated with less IL-5 production from ILC2 cells. Functionally, adoptively transferred pulmonary iEOS from WT but not from C5ar1-/- mice-induced airway hyperresponsiveness (AHR), which was associated with significantly reduced C5ar1-/- iEOS degranulation. Pulmonary iEOS but not rEOS were frequently associated with T cells in lung tissue. After HDM or IL-33 exposure, iEOS but not rEOS were found in mLNs, which were significantly reduced in C5ar1-/- mice. C5ar1-/- iEOS expressed less costimulatory molecules, associated with a decreased potency to drive antigen-specific T cell proliferation and differentiation into memory T cells.We uncovered novel roles for C5aR1 in iEOS trafficking and activation, which affects key aspects of allergic inflammation such as AHR, ILC2, and T cell activation.
Background Mucociliary clearance in the airways is driven by the coordinated beating of ciliated cells. Classical neuromediators such as noradrenalin and acetylcholine increase ciliary beat frequency and thus cilia-driven transport. Despite the fact that the neuromediator serotonin is ciliostimulatory in invertebrates and has been implied in releasing acetylcholine from the airway epithelium, its role in regulating cilia function in vertebrate airways is not established. Methodology/Principal Findings We examined the effects of serotonin on ciliary beat frequency and cilia-driven particle transport in the acutely excised submerged mouse trachea and determined the sources of serotonin in this tissue by immunohistochemistry. Serotonin (100 µM) increased cilary beat frequency (8.9±1.2 Hz to 17.0±2.7 Hz) and particle transport speed (38.9±4.6 µm/s to 83.4±8.3 µm/s) to an extent that was comparable to a supramaximal dose of ATP. The increase in particle transport speed was totally prevented by methysergide (100 µM). Blockade of muscarinic receptors by atropine (1 µM) did not reduce the effect of serotonin, although it was effective in preventing the increase in particle transport speed mediated by muscarine (100 µM). Immunohistochemistry demonstrated serotonin in mast cells pointing towards mast cells and platelets as possible endogenous sources of serotonin. Conclusions/Significance These results indicate that serotonin is a likely endogenous mediator that can increase cilia-driven transport independent from acetylcholine during activation of mast cells and platelets.
Abstract Coronavirus disease 2019 (COVID-19) can damage cerebral small vessels and cause neurological symptoms. Here we describe structural changes in cerebral small vessels of patients with COVID-19 and elucidate potential mechanisms underlying the vascular pathology. In brains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals and animal models, we found an increased number of empty basement membrane tubes, so-called string vessels representing remnants of lost capillaries. We obtained evidence that brain endothelial cells are infected and that the main protease of SARS-CoV-2 (M pro ) cleaves NEMO, the essential modulator of nuclear factor-κB. By ablating NEMO, M pro induces the death of human brain endothelial cells and the occurrence of string vessels in mice. Deletion of receptor-interacting protein kinase (RIPK) 3, a mediator of regulated cell death, blocks the vessel rarefaction and disruption of the blood–brain barrier due to NEMO ablation. Importantly, a pharmacological inhibitor of RIPK signaling prevented the M pro -induced microvascular pathology. Our data suggest RIPK as a potential therapeutic target to treat the neuropathology of COVID-19.
