Myeloid Wnt ligands are required for normal development of dermal lymphatic vasculature

Resident tissue myeloid cells play a role in many aspects of physiology including development of the vascular systems. In the blood vasculature, myeloid cells use VEGFC to promote angiogenesis and can use Wnt ligands to control vascular branching and to promote vascular regression. Here we show that myeloid cells also regulate development of the dermal lymphatic vasculature using Wnt ligands. Using myeloid-specific deletion of the WNT transporter Wntless we show that myeloid Wnt ligands are active at two distinct stages of development of the dermal lymphatics. As lymphatic progenitors are emigrating from the cardinal vein and intersomitic vessels, myeloid Wnt ligands regulate both their numbers and migration distance. Later in lymphatic development, myeloid Wnt ligands regulate proliferation of lymphatic endothelial cells (LEC) and thus control lymphatic vessel caliber. Myeloid-specific deletion of WNT co-receptor Lrp5 or Wnt5a gain-of-function also produce elevated caliber in dermal lymphatic capillaries. These data thus suggest that myeloid cells produce Wnt ligands to regulate lymphatic development and use Wnt pathway co-receptors to regulate the balance of Wnt ligand activity during the macrophage-LEC interaction

Protease-activated receptor 2 activation of myeloid dendritic cells regulates allergic airway inflammation

<p>Abstract</p> <p>Background</p> <p>A common characteristic of allergens is that they contain proteases that can activate protease-activated receptor (PAR-2); however the mechanism by which PAR-2 regulates allergic airway inflammation is unclear.</p> <p>Methods</p> <p>Mice (wild type and PAR-2-deficient) were sensitized using German cockroach (GC) feces (frass), the isolated protease from GC frass, or through adoptive transfer of GC frass-treated bone marrow-derived dendritic cells (BMDC) and measurements of airway inflammation (cellular infiltration, cytokine expression, and mucin production), serum IgE levels and airway hyperresponsiveness (AHR) were assessed. BMDC were cultured, treated with GC frass and assessed for cytokine production. PAR-2 expression on pulmonary mDCs was determined by flow cytometry.</p> <p>Results</p> <p>Exposure to GC frass induced AHR and airway inflammation in wild type mice; however PAR-2-deficient mice had significantly attenuated responses. To directly investigate the role of the protease, we isolated the protease from GC frass and administered the endotoxin-free protease into the airways of mice in the presence of OVA. GC frass proteases were sufficient to promote the development of AHR, serum IgE, and Th2 cytokine production. PAR-2 expression on mDC was upregulated following GC frass exposure, but the presence of a functional PAR-2 did not alter antigen uptake. To determine if PAR-2 activation led to differential cytokine production, we cultured BMDC in the presence of GM-CSF and treated these cells <it>ex vivo </it>with GC frass. PAR-2-deficient BMDC released significantly less IL-6, IL-23 and TNFα compared to BMDC from wild type mice, suggesting PAR-2 activation was important in Th2/Th17 skewing cytokine production. To determine the role for PAR-2 on mDCs on the initiation of allergic airway inflammation, BMDCs from wild type and PAR-2-deficient mice were treated in the presence or absence of GC frass and then adoptively transferred into the airway of wild type mice. Importantly, GC frass-stimulated wild type BMDCs were sufficient to induce AHR and allergic airway inflammation, while GC frass-stimulated PAR-2-deficient BMDC had attenuated responses.</p> <p>Conclusions</p> <p>Together these data suggest an important role for allergen activation of PAR-2 on mDCs in mediating Th2/Th17 cytokine production and allergic airway responses.</p

CD4+CD25+ T cells protect against experimentally induced asthma and alter pulmonary dendritic cell phenotype and function

The role of natural CD4+CD25+ regulatory T (T reg) cells in the control of allergic asthma remains poorly understood. We explore the impact of T reg cell depletion on the allergic response in mice susceptible (A/J) or comparatively resistant (C3H) to the development of allergen-induced airway hyperresponsiveness (AHR). In C3H mice, anti-CD25–mediated T reg cell depletion before house dust mite treatment increased several features of the allergic diathesis (AHR, eosinophilia, and IgE), which was concomitant with elevated T helper type 2 (Th2) cytokine production. In similarly T reg cell–depleted A/J mice, we observed a moderate increase in airway eosinophilia but no effects on AHR, IgE levels, or Th2 cytokine synthesis. As our experiments suggested that T reg cell depletion in C3H mice before sensitization was sufficient to enhance the allergic phenotype, we characterized dendritic cells (DCs) in T reg cell–depleted C3H mice. T reg cell–depleted mice had increased numbers of pulmonary myeloid DCs with elevated expression of major histocompatibility complex class II, CD80, and CD86. Moreover, DCs from T reg cell–depleted mice demonstrated an increased capacity to stimulate T cell proliferation and Th2 cytokine production, which was concomitant with reduced IL-12 expression. These data suggest that resistance to allergen-driven AHR is mediated in part by CD4+CD25+ T reg cell suppression of DC activation and that the absence of this regulatory pathway contributes to susceptibility

Allergen Uptake, Activation, and IL-23 Production by Pulmonary Myeloid DCs Drives Airway Hyperresponsiveness in Asthma-Susceptible Mice

Maladaptive, Th2-polarized inflammatory responses are integral to the pathogenesis of allergic asthma. As regulators of T cell activation, dendritic cells (DCs) are important mediators of allergic asthma, yet the precise signals which render endogenous DCs “pro-asthmatic”, and the extent to which these signals are regulated by the pulmonary environment and host genetics, remains unclear. Comparative phenotypic and functional analysis of pulmonary DC populations in mice susceptible (A/J), or resistant (C3H) to experimental asthma, revealed that susceptibility to airway hyperresponsiveness is associated with preferential myeloid DC (mDC) allergen uptake, and production of Th17-skewing cytokines (IL-6, IL-23), whereas resistance is associated with increased allergen uptake by plasmacytoid DCs. Surprisingly, adoptive transfer of syngeneic HDM-pulsed bone marrow derived mDCs (BMDCs) to the lungs of C3H mice markedly enhanced lung IL-17A production, and rendered them susceptible to allergen-driven airway hyperresponsiveness. Characterization of these BMDCs revealed levels of antigen uptake, and Th17 promoting cytokine production similar to that observed in pulmonary mDCs from susceptible A/J mice. Collectively these data demonstrate that the lung environment present in asthma-resistant mice promotes robust pDC allergen uptake, activation, and limits Th17-skewing cytokine production responsible for driving pathologic T cell responses central to the development of allergen-induced airway hyperresponsiveness

The role of protease-activated receptor-2 on pulmonary neutrophils in the innate immune response to cockroach allergen

Abstract Background Serine proteases in German cockroach (GC) have been shown to mediate allergic airway inflammation through the activation of protease activated receptor (PAR)-2. Neutrophils play an important role in regulating the innate immune response, and are recruited into the airways following GC frass exposure. As such, we investigated the role of PAR-2 in airway neutrophil recruitment, activation and cytokine production following allergen exposure. Methods Wild type and PAR-2-deficient mice were administered a single intratracheal instillation of PBS or GC frass and neutrophil recruitment, expression of PAR-2, CD80, CD86, and MHC class II were assessed by flow cytometry and levels of tumor necrosis factor (TNF)α was assessed by ELISA. Uptake of AlexaFluor 405-labeled GC frass by neutrophils was performed by flow cytometry. Results Neutrophil recruitment in the lung and airways following GC frass exposure was significantly decreased in PAR-2-deficient mice compared to wild type mice. GC frass exposure increased the level of PAR-2 on pulmonary neutrophils and increased numbers of PAR-2-positive neutrophils were found in the lungs; however PAR-2 did not play a role in meditating allergen uptake. Comparing wild type and PAR-2-deficient mice, we found that a single exposure to GC frass increased levels of CD80 and CD86 on pulmonary neutrophils, an effect which was independent of PAR-2 expression. Neutrophils isolated from the whole lungs of naïve PAR-2-deficient mice treated ex vivo with GC frass produced significantly less TNFα than in similarly treated wild type neutrophils. Lastly, neutrophils were isolated from the bronchoalveolar lavage fluid of wild type and PAR-2-deficient mice following a single intratracheal exposure to GC frass. Airway neutrophils from PAR-2-deficient mice released substantially decreased levels of TNFα, suggesting a role for PAR-2 in neutrophil-derived cytokine production. Conclusions Together these data suggest PAR-2 expression can be upregulated on lung neutrophils following allergen exposure and the consequence is altered release of TNFα which could drive the early innate immune response.</p

Distribution and Interaction of Murine Pulmonary Phagocytes in the Naive and Allergic Lung

The division of labor between pulmonary phagocytic subsets [macrophage/monocyte and dendritic cell (DC) subpopulations] has been described at the functional level. However, whether these lung phagocytes also display unique spatial distribution remains unclear. Here, to analyze cellular distribution in lung compartments and contacts between phagocyte subpopulations, we established an immunohistochemistry (IHC)-based method to clearly identify murine lung phagocyte subsets in situ based on differential expression of CD11c, CD11b, MHC-II, Langerin and mPDCA-1. Furthermore, we investigated subset-specific functional differences in antigen uptake and spatial changes upon allergic sensitization. Our staining allowed the distinction between alveolar macrophages (AMs), interstitial macrophage (IM) subpopulations, CD11b+ DC subpopulations, CD103+ DCs, and plasmacytoid DCs (pDCs). We identified interstitial regions between airways and around airways as regions of IM/CD11b+ DC/CD103+ DC clusters, where a subset of IMs (IM2) and CD103+ DCs formed intense contacts that decreased upon allergic sensitization. These data indicate functional interactions between both cell types either in steady state or after antigen encounter affecting the development of allergies or tolerance. Furthermore, we observed major antigen uptake in AMs and IMs rather than DC subpopulations that was not restricted to airways and adjacent areas. This will enable to focus future studies to immunologically relevant cellular interactions and to unravel which cells are tipping the balance between pro-inflammatory immune responses or tolerance

Origin, localization and immunoregulatoy properties of pulmonary phagocytes in allergic asthma

Allergic asthma is a chronic inflammatory disease of the airways that is driven by maladaptive T helper 2 (Th2) and Th17 immune responses against harmless, airborne substances. Pulmonary phagocytes represent the first line of defense in the lung where they constantly sense the local environment for potential threats. They comprise two distinct celltypes, i.e. macrophages and dendritic cells (DC) that differ in their origins and functions. Alveolar macrophages quickly take up most of the inhaled allergens, yet do not deliver their cargo to naïve T cells sampling in draining lymph nodes. In contrast, pulmonary DCs instruct CD4+ T cells develop into Th2 and Th17 effectors, initiating the maladaptive immune responses towards harmless environmental substances observed in allergic individuals. Unraveling the mechanisms underlying this mistaken identity of harmless, airborne substances by innate immune cells is one of the great challenges in asthma research. The identification of different pulmonary DC subsets, their role in antigen uptake, migration to the draining lymph nodes, and their potential to instruct distinct T cell responses has set the stage to unravel this mystery. However, at this point, a detailed understanding of the spatiotemporal resolution of DC subset localization, allergen uptake, and processing, autocrine and paracrine cellular cross-talk, and the humoral factors that define the activation status of DCs is still lacking. In addition to DCs, at least two distinct macrophage populations have been identified in the lung that are either located in the airway/alveolar lumen or in the interstitium. Recent data suggest that such populations can exert either pro or anti-inflammatory functions. Similar to the DC subsets, detailed insights into the individual roles of alveolar and interstitial macrophages during the different phases of asthma development are still missing. Here, we will provide an update on the current understanding of the origin, localization and function of the diverse pulmonary antigen-presenting cell subsets, in particular with regard to the development and regulation of allergic asthma. While most data are from mouse models of experimental asthma, we have also included available human data to judge the translational value of the findings obtained in experimental asthma models