Mouse respiratory tract dendritic cell subsets and the immunological fate of inhaled antigens

It is widely accepted that tissue dendritic cells (DC) function as immune sentinels by alerting T cells to foreign antigen after delivering and presenting it in the draining lymph nodes. Over the last two decades, studies in animal models, particularly rodents, have demonstrated that respiratory tract DC are crucial for the adaptive immune response to inhaled antigen. Indeed, the fate of inhaled antigen is inextricably linked to the function of respiratory tract DC. In this review, we will discuss the characteristics of respiratory tract DC from mice and recent data that may help to explain their role in the fate of inhaled antigen

Attenuation of allergen-induced airway hyperresponsiveness is mediated by airway regulatory T cells

Understanding the mechanisms involved in respiratory tolerance to inhaled allergens could potentially result in improved therapies for asthma and allergic diseases. Airway hyperresponsiveness (AHR) is a major feature of allergic asthma, thus the aim of the current study was to investigate mechanisms underlying suppression of allergen-induced AHR during chronic allergen exposure. Adult BALB/c mice were systemically sensitized with ovalbumin (OVA) in adjuvant and then challenged with a single 3 or 6 wk of OVA aerosols. Airway and parenchymal responses to inhaled methacholine (MCh), inflammatory cell counts, cytokines, OVA-specific IgE and IgG1, parenchymal histology, and numbers of airway CD4+69+ activated and CD4+25+FoxP3+ regulatory T (Treg) cells were assessed 24 h after the final aerosol. Single OVA challenge resulted in AHR, eosinophilia, increased serum OVA-specific IgE, and T helper 2 (Th2) cytokines in bronchoalveolar lavage (BAL) but no difference in numbers of Treg compared with control mice. Three weeks of OVA challenges resulted in suppression of AHR and greater numbers of airway Treg cells and increased transforming growth factor-β1 (TGFβ1) compared with control mice despite the presence of increased eosinophilia, OVA-specific IgE and IgG1, and airway remodeling. Six weeks of OVA challenges restored AHR, whereas airway Treg numbers, TGFβ1, BAL eosinophilia, and Th2 cytokines returned to control levels. Partial in vivo depletion or adoptive transfer of Treg cells restored or inhibited AHR, respectively, but did not affect TGFβ1 or Th2 cytokine production. In conclusion, AHR suppression is mediated by airway Treg cells and potentially via a paracrine induction of TGFβ1 in the airways

Lung homing T-cell generation is dependent on strength and timing of antigen delivery to lymph nodes

Inhaled allergens are known for their immediate and ongoing effects in the respiratory tract (RT). In this report, we track inhaled antigen in normal mice for 7 days and find that while it is cleared from the airways, inhaled antigen persists in peripheral lung tissue and the draining lymph nodes (DLNs). The persistence of antigen led to ongoing presentation in the lymph nodes, but not the lungs, that decreased with time in direct proportion with the frequency of antigen-bearing RT dendritic cells (DCs). There was evidence of functional changes among the antigen-bearing DCs in the lymph nodes, as the expression of CD40, CD80 and CD86 were modulated over the course of 7 days. At the same time, there was a decrease in both CD4 T-cell proliferation in lymph nodes and the generation of recirculating CD4 T cells. However, early presentation of lower doses of inhaled antigen also resulted in a decrease in CD4 T-cell proliferation and recirculation. Thus, T-cell recirculation depends on the strength of stimulus in the DLNs and is produced by a combination of the dose of antigen delivered to the RT, DC migration and co-stimulatory molecule expression. These results provide an important insight into the fate of inhaled antigen in vivo and the influence of persistent antigen presentation on T-cell activation in the lymph nodes

Identification and isolation of rodent respiratory tract dendritic cells

This chapter describes the preparation of respiratory tract tissue from both mice and rats for the isolation of respiratory tract dendritic cells (RTDC). The methods describe in detail the preparation of cells from the respiratory tract tissue of the main conducting airways (representing mucosal populations) and peripheral lung (representing predominantly interstitial populations) in both rodent species. Our research in this area has found that these anatomical sites differ in their composition of antigen-presenting cell (APC) types including RTDC, and that phenotypic and functional differences exist in RTDC isolated from these sites. We predominantly use a flow cytometry-based approach to identify and sort RTDC as this is the most accurate way of isolating RTDC subsets in an environment where many typical dendritic cell surface markers are shared by other APC populations

Airway hyperresponsiveness in mouse models of asthma is associated with activated T cells in the airways

Adoptive transfer of activated T cells has been shown to induce allergic responses in the lung, however, direct physiological evidence of whether these T cells home to the airways is lacking. This study aimed to determine the role of CD4+ T cells in the generation of airway hyperresponsiveness (AHR) in mouse models of asthma. Methods: (1) 129/Sv, C57BL/6 and BALB/c mice were sensitized and challenged with ovalbumin (OVA). AHR, inflammatory cells, serum IgE and IgG1 and the number of CD4+ CD69+ T cells in the trachea and peripheral lung were measured. (2) DO11.10 transgenic T cells that recognize OVAwere transferred to naïve BALB/c recipients. Recipient mice were primed and challenged with OVAand assessed forAHR and serum antibodies. (3) Naïve BALB/c mice were passively sensitized with high titre IgE/IgG1 titre serum, challenged with OVA and assessed for AHR. Results: (1) AHR to inhaled methacholine (MCh) was induced by OVA in BALB/c mice only. This correlated with the presence of CD4+ CD69+ T cells and IgG1 . (2) After 5 OVA challenges naïve BALB/c mice primed with DO11.10 T cells demonstrated AHR (p=0.049) to MCh. (3) Passive transfer of high titre IgE/IgG1 serum did not result in AHR. Conclusions: The presence of AHR in BALB/c mice was linked to the numbers of CD4+ CD69+ T cells and IgG1. Adoptive transfer of T cells that recognize OVA resulted in AHR following challenge suggesting that these T cells traffic to the airway after challenge. This could not be replicated by passively sensitizing mice with high IgE/IgG1 titre serum alone. This study highlighted the potential role of CD4+ T cells in the development of AHR and further studies using this system may be able to dissect the mechanism by which this occurs

Influence of mucosal adjuvants on antigen passage and CD4+ T cell activation during the primary response to airborne allergen

Ag delivery via the nasal route typically induces tolerance or fails to polarize CD4+ T cell responses unless an adjuvant is provided. To better understand this process, we assessed the effects of two mucosal adjuvants, Escherichia coli LPS and cholera toxin (CT), on Ag passage and T cell activation in the draining lymph nodes (DLN) of BALB/c mice following per nasal administration of the model protein allergen, OVA. We found a range of cell types acquired small amounts of fluorescent OVA in the DLN 4 h after per nasal administration. However, this early uptake was eclipsed by a wave of OVA+CD8alpha(low) dendritic cells that accumulated in the DLN over the next 20 h to become the dominant OVA-processing and -presenting population. Both LPS and CT stimulated increases in CD80 and CD86 expression on OVA+CD8alpha(low) DC. LPS also increased the number of OVA+CD8alpha(low) dendritic cells accumulating in the DLN. When the primary T cell response was examined after adoptive transfer of CD4+ T cells from DO11.10 mice, CT and LPS stimulated surprisingly similar effects on T cell activation and proliferation, IL-4 and IFN-gamma priming, and memory T cell production. Despite these similarities, T cell recipients immunized with CT, but not LPS, developed lung eosinophilia upon secondary OVA challenge. Thus, we found no bias within the DLN in Ag handling or the primary T cell response associated with the eventual Th2 polarization induced by CT, and suggest that additional tissue-specific factors influence the development of allergic disease in the airways

Anatomical location determines the distribution and function of dendritic cells and other APCs in the respiratory tract.

APCs, including dendritic cells (DC), are central to Ag surveillance in the respiratory tract (RT). Research in this area is dominated by mouse studies on purportedly representative RT-APC populations derived from whole-lung digests, comprising mainly parenchymal tissue. Our recent rat studies identified major functional differences between DC populations from airway mucosal vs parenchymal tissue, thus seriously questioning the validity of this approach. We addressed this issue for the first time in the mouse by separately characterizing RT-APC populations from these two different RT compartments. CD11c(high) myeloid DC (mDC) and B cells were common to both locations, whereas a short-lived CD11c(neg) mDC was unique to airway mucosa and long-lived CD11c(high) macrophage and rapid-turnover multipotential precursor populations were predominantly confined to the lung parenchyma. Airway mucosal mDC were more endocytic and presented peptide to naive CD4+ T cells more efficiently than their lung counterparts. However, mDC from neither site could present whole protein without further maturation in vitro, or following trafficking to lymph nodes in vivo, indicating a novel mechanism whereby RT-DC function is regulated at the level of protein processing but not peptide loading for naive T cell activation

Airway hyperresponsiveness is associated with activated CD4+ T cells in the airways

It is widely accepted that atopic asthma depends on an allergic response in the airway, yet the immune mechanisms that underlie the development of airway hyperresponsiveness (AHR) are poorly understood. Mouse models of asthma have been developed to study the pathobiology of this disease, but there is considerable strain variation in the induction of allergic disease and AHR. The aim of this study was to compare the development of AHR in BALB/c, 129/Sv, and C57BL/6 mice after sensitization and challenge with ovalbumin (OVA). AHR to methacholine was measured using a modification of the forced oscillation technique in anesthetized, tracheostomized mice to distinguish between airway and parenchymal responses. Whereas all strains showed signs of allergic sensitization, BALB/c was the only strain to develop AHR, which was associated with the highest number of activated (CD69+) CD4+ T cells in the airway wall and the highest levels of circulating OVA-specific IgG1. AHR did not correlate with total or antigen-specific IgE. We assessed the relative contribution of CD4+ T cells and specific IgG1 to the development of AHR in BALB/c mice using adoptive transfer of OVA-specific CD4+ T cells from DO11.10 mice. AHR developed in these mice in a progressive fashion following multiple OVA challenges. There was no evidence that antigen-specific antibody had a synergistic effect in this model, and we concluded that the number of antigen-specific T cells activated and recruited to the airway wall was crucial for development of AHR

Allergic airways disease develops after an increase in allergen capture and processing in the airway mucosa

Airway mucosal dendritic cells (AMDC) and other airway APCs continuously sample inhaled Ags and regulate the nature of any resulting T cell-mediated immune response. Although immunity develops to harmful pathogens, tolerance arises to nonpathogenic Ags in healthy individuals. This homeostasis is thought to be disrupted in allergic respiratory disorders such as allergic asthma, such that a potentially damaging Th2-biased, CD4(+) T cell-mediated inflammatory response develops against intrinsically nonpathogenic allergens. Using a mouse model of experimental allergic airways disease (EAAD), we have investigated the functional changes occurring in AMDC and other airway APC populations during disease onset. Onset of EAAD was characterized by early and transient activation of airway CD4(+) T cells coinciding with up-regulation of CD40 expression exclusively on CD11b(-) AMDC. Concurrent enhanced allergen uptake and processing occurred within all airway APC populations, including B cells, macrophages, and both CD11b(+) and CD11b(-) AMDC subsets. Immune serum transfer into naive animals recapitulated the enhanced allergen uptake observed in airway APC populations and mediated activation of naive allergen-specific, airway CD4(+) T cells following inhaled allergen challenge. These data suggest that the onset of EAAD is initiated by enhanced allergen capture and processing by a number of airway APC populations and that allergen-specific Igs play a role in the conversion of normally quiescent AMDC subsets into those capable of inducing airway CD4(+) T cell activation