Neuropeptides influence airway dendritic cell behavior

The airway mucosal epithelium permanently faces airborne particles. A network of immune cells patrols at this frontier to the environmental surface. The interplay of immune cells is orchestrated by different mediators. In the current study we investigated whether neuropeptides can alter key features of dendritic cells (DC) such as movement behavior and phagocytosis capacity. With a two-photon microscopic time-lapse analysis of DC in the airways of ex vivo vital lung sections of CD11c-EYFP transgenic mice we focused on the influence of neuropeptides on DC. Additionally, with a confocal microscopic approach and by means of particles becoming fluorescent in the phagolysosomal milieu we determined the phagocytosis capacity of CD11c+ cells. Neuronal irritation here mimicked by electrical field stimulation (EFS) leads to an unspecific release of several neuropeptides in nerves. After EFS of vital lung slices, airway DC showed an increased motility. In subsequent experiments this effect could be reproduced by specific application of the neuropeptide calcitonin gene-related peptide (CGRP). The EFS-mediated effect could partially be blocked by pre-treatment with the neuropeptide receptor antagonist CGRP8-37. In contrast, the application of the neuropeptide vasoactive intestinal peptide (VIP) led to a decrease of airway DC motility. Additionally, phagocytosis capacity of bone marrow-derived and whole lung CD11c+ cells could be negatively affected by neuropeptides CGRP, VIP, and Substance P. We then correlated these data with the in vivo situation by analyzing DC motility in two different OVA asthma models. Both in the acute and prolonged OVA asthma model we could determine altered neuropeptide amounts in the airways and DC motility. In summary, our data suggest that neuropeptides alter key features motility and phagocytosis of mouse airway DC and therefore might contribute to the pathophysiology of asthma. This work was funded by the SFB 587 B

Spatiotemporal and functional behavior of airway dendritic cells visualized by two-photon microscopy

Airway mucosal dendritic cells (DCs), located beneath the epithelium of the conducting airways, are believed to be specialized for immunosurveillance via sampling of antigens from the airway luminal surface. However, the dynamics of airway DC activity has not yet been visualized. We used two-photon microscopy to illuminate the endogenous mucosal DC network in the airways of mice. To characterize DC behavior, we used lung section preparations and an intravital microscopic approach. DCs displayed a heterogeneous movement pattern according to their localization within the airway mucosa: sessile intraepithelial DCs with a dendritiform shape exhibited active probing movements and occasionally formed transepithelial extensions into the airway lumen. In contrast, DCs within the deeper layers of the mucosal tissue migrated fast in an amoeboid manner, without probing movements, and slowed down after aeroallergen challenge. Strikingly, neither of these two mucosal DC populations ingested fluorescently labeled antigens after antigen administration to the airways in the steady state, in contrast to alveolar macrophage/DC populations in the lung periphery. Our results provide a first description of the dynamic behavior of airway mucosal DCs, with their exact role in antigen sampling remaining unclear

Dissemination of Persistent Intestinal Bacteria via the Mesenteric Lymph Nodes Causes Typhoid Relapse▿

Enteric pathogens can cause relapsing infections in a proportion of treated patients, but greater understanding of this phenomenon is hindered by the lack of appropriate animal models. We report here a robust animal model of relapsing primary typhoid that initiates after apparently successful antibiotic treatment of susceptible mice. Four days of enrofloxacin treatment were sufficient to reduce bacterial loads below detectable levels in all major organs, and mice appeared otherwise healthy. However, any interruption of further antibiotic therapy allowed renewed fecal shedding and renewed bacterial growth in systemic tissues to occur, and mice eventually succumbed to relapsing infection. In vivo imaging of luminescent Salmonella identified the mesenteric lymph nodes (MLNs) as a major reservoir of relapsing infection. A magnetic-bead enrichment strategy isolated MLN-resident CD11b+ Gr-1− monocytes associated with low numbers of persistent Salmonella. However, the removal of MLNs increased the severity of typhoid relapse, demonstrating that this organ serves as a protective filter to restrain the dissemination of bacteria during antibiotic therapy. Together, these data describe a robust animal model of typhoid relapse and identify an important intestinal phagocyte subset involved in protection against the systemic spread of enteric infection

Mesenteric Lymph Nodes Confine Dendritic Cell-Mediated Dissemination of Salmonella enterica Serovar Typhimurium and Limit Systemic Disease in Mice▿

In humans with typhoid fever or in mouse strains susceptible to Salmonella enterica serovar Typhimurium (S. Typhimurium) infection, bacteria gain access to extraintestinal tissues, causing severe systemic disease. Here we show that in the gut-draining mesenteric lymph nodes (MLN), the majority of S. Typhimurium-carrying cells show dendritic-cell (DC) morphology and express the DC marker CD11c, indicating that S. Typhimurium bacteria are transported to the MLN by migratory DCs. In vivo FLT-3L-induced expansion of DCs, as well as stimulation of DC migration by Toll-like receptor agonists, results in increased numbers of S. Typhimurium bacteria reaching the MLN. Conversely, genetically impaired DC migration in chemokine receptor CCR7-deficient mice reduces the number of S. Typhimurium bacteria reaching the MLN. This indicates that transport of S. Typhimurium from the intestine into the MLN is limited by the number of migratory DCs carrying S. Typhimurium bacteria. In contrast, modulation of DC migration does not affect the number of S. Typhimurium bacteria reaching systemic tissues, indicating that DC-bound transport of S. Typhimurium does not substantially contribute to systemic S. Typhimurium infection. Surgical removal of the MLN results in increased numbers of S. Typhimurium bacteria reaching systemic sites early after infection, thereby rendering otherwise resistant mice susceptible to fatal systemic disease development. This suggests that the MLN provide a vital barrier shielding systemic compartments from DC-mediated dissemination of S. Typhimurium. Thus, confinement of S. Typhimurium in gut-associated lymphoid tissue and MLN delays massive extraintestinal dissemination and at the same time allows for the establishment of protective adaptive immune responses

Motility of neuropeptide-treated CD11c⁺ cells.

<p>Mean velocities of lung CD11c⁺ cells and airway DC five minutes after neuropeptide treatment. Groups as indicated. For antagonistic treatment, slices were pre-treated with CGRP_8–37 five minutes prior to EFS and analyzed 1 h after EFS. Each dot represents the mean velocity of an individual cell; black horizontal bars indicate the medians. Data of living lung slices and <i>in vitro</i>-generated BMDC show a pool of three independent experiments for each group. Mann Whitney statistical test; ns not significant; * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001.</p

Determination of neuropeptide amounts and of DC motility in mouse airways in an acute and prolonged model of allergic airway inflammation.

<p>(A) Representative confocal <i>z</i>-stack projections of mouse airways stained as whole-mounts against the pan-neuronal marker PGP9.5 (blue) and neuropeptides SP or CGRP (yellow), respectively. Airways of control-treated and asthmatic mice were analyzed (acute model: i.p. booster on days 14 and 21, challenge and analysis on day 27; prolonged protocol: i.p. booster on days 14 and 21, two additional challenges on days 27 and 28, final challenge on day 35, analysis day 36). The total area of fluorescence signals of maximum intensity projections of <i>z</i>-stack images was determined by using Imaris and ImageJ software. (B) The total area of neuropeptide fluorescence signal (SP or CGRP signal) was set into relation to the area of all nerves (PGP9.5 signal). This ratio was determined for six regions of each airway. Per group three mice donating one airway each were analyzed. Unpaired statistical <i>t</i>-test; outliers were determined by Grubb’s test; ns not significant; * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001. Scale: 40 µm. (C) Mean velocities of airway DC were determined both, in an acute OVA asthma model and in a model using a prolonged protocol and in their respective control groups (ctrl) as indicated. Each dot represents the mean velocity of an individual cell; black horizontal bars indicate the medians. Data of living lung slices show a pool of three (acute) to four (prolonged) independent experiments. Mann-Whitney statistical test; ns not significant; * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001.</p

Characterization of CD11c⁺ cells in mouse main bronchi.

<p>Mouse airways of the CD11c-EYFP reporter mouse (CD11c in yellow) or of the CX₃CR1^+/<i>gfp</i> transgenic mouse (CX₃CR1 in yellow) were stained as whole-mounts against MHC-II (red), F4/80 (red) or CD68 (red) as indicated. Airway epithelium stained with cytokeratin (Ck) is shown in grey. Left column shows merges of all acquired channels. Other columns show according single channel images. Image projections were generated with Imaris (Bitplane) from raw confocal <i>z</i>-stacks. Scale: 40 µm.</p

Phagocytosis capacity of CD11c⁺ cells after neuropeptide treatment.

<p>(A) Representative confocal images of <i>in vitro</i>-cultured BMDC after different treatments as indicated. Upper panel shows a merge of red fluorescence of phagocytosed <i>E. coli</i> pHrodo particles and transmitted light channel to visualize all cells. Lower panel shows only the red fluorescence according to images of the upper panel. Lower panel images were used to determine the total area of red fluorescence in each image with ImageJ software. Scale: 40 µm. (B) Graph of phagocytosis indices (PI) of BMDC and magnetically sorted total lung CD11c⁺ cells. Cells were neuropeptide or control treated for five minutes. <i>E. coli</i> pHrodo particles were then applied for 2 h. Beforehand a fraction of cells was triggered over night with LPS (three independent experiments). To inhibit phagocytosis, another fraction of cells was treated with Cytochalasin D (one experiment for BMDC, two experiments for whole lung CD11c⁺ cells). Neuropeptide-treated groups (0.1 nM VIP, 1 µM SP, 1 nM CGRP) were compared to a water-treated group (ctrl) (four independent experiments for BMDC and two independent experiments for whole lung CD11c⁺ cells). Each dot represents PI of one image. Black horizontal bars indicate the means. Per well two images were acquired. Unpaired statistical <i>t</i>-test; ns not significant; * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001.</p