Lung-draining Lymph Node Cells Research Articles

Signs of Dendritic Cell Activation in the Lungs of Mice Exposed to Aerosols of Flavored Vaping Liquid

Abstract Rationale: Vaping products contain numerous flavor compounds that are known immunological sensitizers. Sensitization to various antigens can be responsible for the onset of serious respiratory diseases, including hypersensivity pneumonitis. However, respiratory tract sensitization and pathological consequences from exposure to aerosols of flavored vaping liquids need to be thoroughly investigated. Methods: Female BALB/c mice were exposed to laboratory made nicotine-free vaping liquid containing a mixture of 1% citral (lemon), 1% cinnamaldehyde (cinnamon), 1% dihydrocoumarin (coconut) and 1% vanillin (vanilla) solubilized in propylene glycol and glycerol (50:50). Mice were exposed 2h/day for 4 days to vaping or room air. Bronchoalveolar lavage was performed to assess total and differential cell counts, and flow cytometry was performed on pulmonary tissue and lung-draining lymph nodes to assess immune cell populations. Results: After 4 days of daily exposure, we found a significant increase in bronchoalveolar lavage immune cells in mice exposed to aerosols from the flavored vaping liquid. We also observed a significant increase in pulmonary conventional dendritic cells (cDCs) as well as their expression of the activation markers CD40, CD86 and MHC-II. This activation is mainly observed on type 2 cDCs. In the lung-draining lymph nodes, flavored vaping liquid lead to a significant increase in total immune cells (CD45+ cells). Conclusions: Pulmonary conventional dendritic cells activation and the increase in total immune cells in lung-draining lymph nodes caused by exposure to aerosols from flavored vaping liquid suggest the early initiation of immunological sensitization processes in the lungs. Fonds de recherche du Québec – Santé, Ministère de la Santé et des Services sociaux, Fondation de l’Institut de cardiologie et de pneumologie de Québec, Réseau de recherche en santé respiratoire du Québec.

Immune-Checkpoint Expression on CD4, CD8 and NK Cells in Blood, Bronchoalveolar Lavage and Lymph Nodes of Sarcoidosis.

BackgroundSarcoidosis features non-necrotizing granulomas consisting mainly of activated CD4-lymphocytes. T-cell activation is regulated by immune checkpoint (IC) molecules. The present study aimed to compare IC expression on CD4, CD8 and NK cells from peripheral, alveolar and lung‐draining lymph node (LLN) samples of sarcoidosis patients.MethodsFlow-cytometry analysis was performed to detect IC molecules and a regression decision tree model was constructed to investigate potential binary classifiers for sarcoidosis diagnosis as well as for the IC distribution.ResultsFourteen patients (7 females) were consecutively recruited in the study; all enrolled patients showed hilo-mediastinal lymph node enlargement and lung parenchyma involvement with chest X-rays and high resolution computed tomography. CD4+PD1+ and CD8+PD1+ were higher in bronchoalveolar lavage (BAL) than in LLN (p = 0.0159 and p = 0.0439, respectively). CD4+ T-cell immunoglobulin and ITIM domain (TIGIT)+ were higher in BAL than in peripheral blood mononuclear cells (PBMCs) (p = 0.0239), while CD8+TIGIT+ were higher in PBMC than in BAL (p = 0.0386). CD56+TIGIT+ were higher in LLN than in PBMC (p = 0.0126). The decision-tree model showed the best clustering cells of PBMC, BAL and LLN: CD56, CD4/CD8 and CD4+TIGIT+ cells. Considering patients and controls, the best subset was CD4+CTLA-4+.ConclusionHigh expression of PD1 and TIGIT on T cells in BAL, as well as CTLA-4 and TIGIT on T cells in LLN, suggest that inhibition of these molecules could be a therapeutic strategy for avoiding the development of chronic inflammation and tissue damage in sarcoidosis patients.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1007/s40291-022-00596-0.

Lung-resident memory B cells established after pulmonary influenza infection display distinct transcriptional and phenotypic profiles.

Recent studies have established that memory B cells, largely thought to be circulatory in the blood, can take up long-term residency in inflamed tissues, analogous to widely described tissue-resident T cells. The dynamics of recruitment and retention of memory B cells to tissues and their immunological purpose remains unclear. Here, we characterized tissue-resident memory B cells (BRM) that are stably maintained in the lungs of mice after pulmonary influenza infection. Influenza-specific BRM were localized within inducible bronchus-associated lymphoid tissues (iBALTs) and displayed transcriptional signatures distinct from classical memory B cells in the blood or spleen while showing partial overlap with memory B cells in lung-draining lymph nodes. We identified lung-resident markers, including elevated expression of CXCR3, CCR6, and CD69, on hemagglutinin (HA)- and nucleoprotein (NP)-specific lung BRM. We found that CCR6 facilitates increased recruitment and/or retention of BRM in lungs and differentiation into antibody-secreting cells upon recall. Although expression of CXCR3 and CCR6 was comparable in total and influenza-specific memory B cells isolated across tissues of human donors, CD69 expression was higher in memory B cells from lung and draining lymph nodes of human organ donors relative to splenic and PBMC-derived populations, indicating that mechanisms underpinning BRM localization may be evolutionarily conserved. Last, we demonstrate that human memory B cells in lungs are transcriptionally distinct to populations in lung-draining lymph nodes or PBMCs. These data suggest that BRM may constitute a discrete component of B cell immunity, positioned at the lung mucosa for rapid humoral response against respiratory viral infections.

Proinflammatory effects of environmental cadmium boost resistance to opportunistic pathogen Aspergillus fumigatus: Implications for sustained low-level pulmonary inflammation?

Cadmium (Cd) is one of the most toxic environmental heavy metals to which the general population is exposed mainly via the oral route. Owing to its immunomodulatory potential, orally acquired Cd affects antimicrobial immune defense in several organs, including the lungs. While there are data concerning Cd and viral and bacterial pulmonary infections, effects on fungal infections are not studied yet. In the present study, the effect of the Cd (5 mg/L for 30 days, in drinking water, the average daily Cd intake 0.641 ± 0.089 mg/kg) on the immune response of rats to pulmonary A. fumigatus infection was examined. Data obtained showed that orally acquired cadmium does not affect the elimination of the fungus in immunocompetent rats owing to the preservation of some aspects of innate immune responses (lung leukocyte infiltration and NBT reduction) and an increase in other (increased numbers of mucus-producing goblet cells, MPO release). Cd does not affect an IFN-γ response in lung leukocytes during the infection (despite suppression of cytokine production in cells of lung-draining lymph nodes), while it stimulates IL-17 and suppresses IL-10 response to the fungus. As a result, the elimination of the fungus occurs in a milieu with the prevailing proinflammatory response in Cd-exposed animals that preserved fungal elimination from the lungs, though with more intense injury to the lung tissue. Therefore, the proinflammatory microenvironment in the lungs created by Cd that sustains inflammatory/immune response to the fungus to which humans are exposed for a lifetime, raises a concern of orally acquired Cd as a risk factor for the development of chronic low-grade pulmonary inflammation.

γ-herpesvirus latency attenuates Mycobacterium tuberculosis infection in mice

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a bacterial pathogen which is transmitted via aerosol and establishes a chronic lung infection. In naïve hosts, Mtb grows for several weeks without being restricted by IFNγ-producing T cells, which eventually accumulate and limit Mtb dissemination. In this study, we used a mouse model of Mtb/γ-herpesvirus (γHV) coinfection to test the hypothesis that latent γHV infection alters host resistance to Mtb. γHVs are DNA viruses which elicit a polyclonal T cell response and attenuate some acute bacterial pathogens in mice; whether γHVs modulate infection with Mtb is unknown. Here, mice harboring latent mouse gammaherpesvirus 68 (MHV68)—a γHV genetically and biologically related to human Epstein Barr virus (EBV)—were infected via aerosol with a low dose of virulent Mtb. Mtb burdens and IFNγ+ T cell frequencies in mice with latent MHV68 (MHV68POS mice) were subsequently measured and compared to control mice that did not harbor latent MHV68 (MHV68NEG mice). Relative to MHV68NEG controls, MHV68POS mice more effectively limited Mtb growth and dissemination, and had higher frequencies of CD4+IFNγ+ cells in lung-draining lymph nodes. Collectively, our results support a model wherein latent γHV confers moderate protection against subsequent Mtb infection.

Enhancement of antigen-specific CD4+ and CD8+ T cell responses using a self-assembled biologic nanolipoprotein particle vaccine

To address the need for vaccine platforms that induce robust cell-mediated immunity, we investigated the potential of utilizing self-assembling biologic nanolipoprotein particles (NLPs) as an antigen and adjuvant delivery system to induce antigen-specific murine T cell responses. We utilized OT-I and OT-II TCR-transgenic mice to investigate the effects of NLP-mediated delivery of the model antigen ovalbumin (OVA) on T cell activation. Delivery of OVA with the TLR4 agonist monophosphoryl lipid A (MPLA) in the context of NLPs significantly enhanced the activation of both CD4+ and CD8+ T cells in vitro compared to co-administration of free OVA and MPLA. Upon intranasal immunization of mice harboring TCR-transgenic cells, NLPs enhanced the adjuvant effects of MPLA and the in vivo delivery of OVA, leading to significantly increased expansion of CD4+ and CD8+ T cells in lung-draining lymph nodes. Therefore, NLPs are a promising vaccine platform for inducing T cell responses following intranasal administration.

Culture supernatant of adipose stem cells can ameliorate allergic airway inflammation via recruitment of CD4+CD25+Foxp3 T cells

BackgroundIn a previous study, we demonstrated that intravenous administration of adipose tissue stem cells (ASCs) could significantly reduce allergic symptoms and suppress eosinophilic inflammation.MethodsTo evaluate the secretome of ASCs, we administrated culture supernatant of ASCs (ASC sup, which contains the ASC secretome) and uncultured fresh medium (con sup) into a mouse model of allergic airway inflammation. Subsequently we observed the mice for signs of inflammation and investigated Th1-, Th2-, and Treg-related cytokine levels as well as recruitment of Treg cells into the airway.ResultsWe found that ASC sup could ameliorate allergic airway inflammation in this model; the value of airway hyperresponsiveness, and the occurrence of inflammatory cell infiltration in the lung, as well as the number of eosinophils, and goblet cells in the lung epithelium were all significantly decreased by ASC sup treatment. In addition, ASC sup treatment significantly decreased the levels of IL-4, IL-5, and IL-13 in the bronchial alveolar lavage fluid and in culture medium of lung-draining lymph node cells of the animal model of acute asthma. We detected numerous CTLA-4 and Foxp3-expressing cells in the lung after ASC sup treatment. ASC sup was found to have a higher concentration of IL-10 and TGF-β compared to con sup.ConclusionsStem cells have powerful potential for therapeutic functions in various diseases, but they also have many drawbacks. In this study, we found strong immunosuppressive ability of ASC sup in an allergic airway mouse model. It may be possible to use ASC sup for treatment of many immunological diseases in the near future.

IL-23 secreted by bronchial epithelial cells contributes to allergic sensitization in asthma model: role of IL-23 secreted by bronchial epithelial cells.

IL-23 has been postulated to be a critical mediator contributing to various inflammatory diseases. Dermatophagoides pteronyssinus (Der p) is one of the most common inhalant allergens. However, the role of IL-23 in Der p-induced mouse asthma model is not well understood, particularly with regard to the development of allergic sensitization in the airways. The objective of this study was to evaluate roles of IL-23 in Der p sensitization and asthma development. BALB/c mice were repeatedly administered Der p intranasally to develop Der p allergic sensitization and asthma. After Der p local administration, changes in IL-23 expression were examined in lung tissues and primary epithelial cells. Anti-IL-23p19 antibody was given during the Der p sensitization period, and its effects were examined. Effects of anti-IL-23p19 antibody at bronchial epithelial levels were also examined in vitro. The expression of IL-23 at bronchial epithelial layers was increased after Der p local administration in mouse. In Der p-induced mouse models, anti-IL-23p19 antibody treatment during allergen sensitization significantly diminished Der p allergic sensitization and several features of allergic asthma including the production of Th2 cytokines and the population of type 2 innate lymphoid cells in lungs. The activation of dendritic cells in lung-draining lymph nodes was also reduced by anti-IL-23 treatment. In murine lung alveolar type II-like epithelial cell line (MLE-12) cells, IL-23 blockade prevented cytokine responses to Der p stimulation, such as IL-1α, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-33, and also bone marrow-derived dendritic cell activation. In conclusion, IL-23 is another important bronchial epithelial cell-driven cytokine which may contribute to the development of house dust mite allergic sensitization and asthma.

Cholesterol-sensing liver X receptors stimulate Th2-driven allergic eosinophilic asthma in mice.

IntroductionLiver X receptors (LXRs) are nuclear receptors that function as cholesterol sensors and regulate cholesterol homeostasis. High cholesterol has been recognized as a risk factor in asthma; however, the mechanism of this linkage is not known.MethodsTo explore the importance of cholesterol homeostasis for asthma, we investigated the contribution of LXR activity in an ovalbumin‐ and a house dust mite‐driven eosinophilic asthma mouse model.ResultsIn both models, airway inflammation, airway hyper‐reactivity, and goblet cell hyperplasia were reduced in mice deficient for both LXRα and LXRβ isoforms (LXRα−/−β−/−) as compared to wild‐type mice. Inversely, treatment with the LXR agonist GW3965 showed increased eosinophilic airway inflammation. LXR activity contributed to airway inflammation through promotion of type 2 cytokine production as LXRα−/−β−/− mice showed strongly reduced protein levels of IL‐5 and IL‐13 in the lungs as well as reduced expression of these cytokines by CD4+ lung cells and lung‐draining lymph node cells. In line herewith, LXR activation resulted in increased type 2 cytokine production by the lung‐draining lymph node cells.ConclusionsIn conclusion, our study demonstrates that the cholesterol regulator LXR acts as a positive regulator of eosinophilic asthma in mice, contributing to airway inflammation through regulation of type 2 cytokine production.

Invariant Natural Killer T Cells Promote T Cell Immunity by Modulating the Function of Lung Dendritic Cells during Chlamydia pneumoniae Infection

In this study, we examined the effect of invariant natural killer T (iNKT) cells on the function of lung dendritic cells (LDCs) in eliciting protective immunity against Chlamydia pneumoniae (Cpn) lung infection. We employed a combination of approaches including the use of iNKT cell-deficient, Jα18-knockout (KO) mice and LDC adoptive transfer. We found that iNKT cells significantly altered the number, phenotype and cytokine profile of LDCs following infection. Furthermore, coculture of T cells with LDCs from Cpn-infected wild-type (WT) and KO mice induced type-1 and type-2 responses, respectively. More importantly, upon adoptive transfer, LDCs from Cpn-infected WT mice (WT-LDCs) conferred protective immunity, whereas LDCs from KO mice (KO-LDCs) increased the severity of disease after challenge infection. Further cytokine analyses of the lung tissues and lung-draining lymph node cells showed that KO-LDC-recipient mice exhibited a type-2 cytokine production pattern, while WT-LDC recipients exhibited a type-1 cytokine profile. Taken together, our results provide in vivo evidence that iNKT cells play a critical role in modulating LDC function to generate protective T-cell immunity, particularly in a clinically relevant intracellular bacterial infection.

Dectin-2 Regulates the Effector Phase of House Dust Mite–Elicited Pulmonary Inflammation Independently from Its Role in Sensitization

The myeloid C-type lectin receptor Dectin-2 directs the generation of Th2 and Th17 immune responses to the house dust mite Dermatophagoides farinae through the generation of cysteinyl leukotrienes and proinflammatory cytokines, respectively, but a role for Dectin-2 in effector phase responses has not been described. In this study, we demonstrate that administration of the Dectin-2 mAb solely at the time of D. farinae challenge abrogated eosinophilic and neutrophilic inflammation in the bronchoalveolar lavage fluid and Th1, Th2, and Th17 inflammation in the lung of previously sensitized mice. Furthermore, Dectin-2 null mice (Clec4n(-/-)) sensitized with the adoptive transfer of D. farinae-pulsed wild-type (WT) bone marrow-derived dendritic cells (DCs) also had less D. farinae-elicited pulmonary inflammation, supporting an effector function for Dectin-2. The protection from pulmonary inflammation seen with the Dectin-2 mAb or in Clec4n(-/-) mice was associated with little or no reduction in lung-draining lymph node cells or their cytokine production and with no reduction in serum IgE. WT and Clec4n(-/-) mice recipients, sensitized with D. farinae-pulsed WT bone marrow-derived DCs, had comparable levels of D. farinae-elicited IL-6, IL-23, TNF-α, and cysteinyl leukotrienes in the lung. By contrast, D. farinae-elicited CCL4 and CCL8 production from pulmonary CD11c(+)CD11b(+)Ly6C(+) and CD11c(+)CD11b(+)Ly6C(-)CD64(+) monocyte-derived DCs was reduced in Clec4n(-/-) recipients. Addition of CCL8 at the time of D. farinae challenge abrogated the protection from eosinophilic, neutrophilic, and Th2 pulmonary inflammation seen in Clec4n(-/-) recipients. Taken together, these results reveal that Dectin-2 regulates monocyte-derived DC function in the pulmonary microenvironment at D. farinae challenge to promote the local inflammatory response.

Size-Dependent Uptake of Particles by Pulmonary Antigen-Presenting Cell Populations and Trafficking to Regional Lymph Nodes

The respiratory tract is an attractive target organ for novel diagnostic and therapeutic applications with nano-sized carriers, but their immune effects and interactions with key resident antigen-presenting cells (APCs) such as dendritic cells (DCs) and alveolar macrophages (AMs) in different anatomical compartments remain poorly understood. Polystyrene particles ranging from 20 nm to 1,000 nm were instilled intranasally in BALB/c mice, and their interactions with APC populations in airways, lung parenchyma, and lung-draining lymph nodes (LDLNs) were examined after 2 and 24 hours by flow cytometry and confocal microscopy. In the main conducting airways and lung parenchyma, DC subpopulations preferentially captured 20-nm particles, compared with 1,000-nm particles that were transported to the LDLNs by migratory CD11blow DCs and that were observed in close proximity to CD3⁺ T cells. Generally, the uptake of particles increased the expression of CD40 and CD86 in all DC populations, independent of particle size, whereas 20-nm particles induced enhanced antigen presentation to CD4⁺ T cells in LDLNs in vivo. Despite measurable uptake by DCs, the majority of particles were taken up by AMs, irrespective of size. Confocal microscopy and FACS analysis showed few particles in the main conducting airways, but a homogeneous distribution of all particle sizes was evident in the lung parenchyma, mostly confined to AMs. Particulate size as a key parameter determining uptake and trafficking therefore determines the fate of inhaled particulates, and this may have important consequences in the development of novel carriers for pulmonary diagnostic or therapeutic applications.

Mannose-Capped Lipoarabinomannan from Mycobacterium tuberculosis Preferentially Inhibits Sphingosine-1-Phosphate–Induced Migration of Th1 Cells

Chemokine receptor cross-desensitization provides an important mechanism to regulate immune cell recruitment at sites of inflammation. We previously reported that the mycobacterial cell wall glycophospholipid mannose-capped lipoarabinomannan (ManLAM) could induce human peripheral blood T cell chemotaxis. Therefore, we examined the ability of ManLAM to desensitize T cells to other chemoattractants as a potential mechanism for impaired T cell homing and delayed lung recruitment during mycobacterial infection. We found that ManLAM pretreatment inhibited in vitro migration of naive human or mouse T cells to the lymph node egress signal sphingosine-1-phosphate (S1P). Intratracheal administration of ManLAM in mice resulted in significant increases in T cells, primarily CCR5(+) (Th1) cells, in lung-draining lymph nodes. To investigate the selective CCR5 effect, mouse T cells were differentiated into Th1 or Th2 populations in vitro, and their ability to migrate to S1P with or without ManLAM pretreatment was analyzed. ManLAM pretreatment of Th1 populations inhibited S1P-induced migration but had no effect on Th2 cell S1P-directed migration, suggesting a differential effect by S1P on the two subsets. The PI3K/AKT inhibitor Ly294002 inhibited S1P-directed migration by Th1 cells, whereas the ERK inhibitor U0126 inhibited Th2 cell S1P-directed migration. These observations demonstrate that S1P-induced migratory responses in Th1 and Th2 lymphocytes occurs via different signaling pathways and suggests further that the production of ManLAM during Mycobacterium tuberculosis infection may function to sequester Th1 cells in lung-draining lymph nodes, thereby delaying their recruitment to the lung.

Increased Th2 Cytokine Secretion, Eosinophilic Airway Inflammation, and Airway Hyperresponsiveness in Neurturin-Deficient Mice

Neurotrophins such as nerve growth factor and brain-derived neurotrophic factor have been described to be involved in the pathogenesis of asthma. Neurturin (NTN), another neurotrophin from the glial cell line-derived neurotrophic factor family, was shown to be produced by human immune cells: monocytes, B cells, and T cells. Furthermore, it was previously described that the secretion of inflammatory cytokines was dramatically stimulated in NTN knockout (NTN(-/-)) mice. NTN is structurally similar to TGF-β, a protective cytokine in airway inflammation. This study investigates the implication of NTN in a model of allergic airway inflammation using NTN(-/-) mice. The bronchial inflammatory response of OVA-sensitized NTN(-/-) mice was compared with wild-type mice. Airway inflammation, Th2 cytokines, and airway hyperresponsiveness (AHR) were examined. NTN(-/-) mice showed an increase of OVA-specific serum IgE and a pronounced worsening of inflammatory features. Eosinophil number and IL-4 and IL-5 concentration in the bronchoalveolar lavage fluid and lung tissue were increased. In parallel, Th2 cytokine secretion of lung draining lymph node cells was also augmented when stimulated by OVA in vitro. Furthermore, AHR was markedly enhanced in NTN(-/-) mice after sensitization and challenge when compared with wild-type mice. Administration of NTN before challenge with OVA partially rescues the phenotype of NTN(-/-) mice. These findings provide evidence for a dampening role of NTN on allergic inflammation and AHR in a murine model of asthma.

High‐fat feeding redirects cytokine responses and decreases allergic airway eosinophilia

Dietary fat intake has been associated with obesity and obesity in its turn with attenuated airway function and asthma, but it is unclear whether or how high-fat intake per se alters immune function relevant to development of allergic asthma. To use a non-obese mouse model of mild to moderate allergic asthma to compare effects of high-fat with isocaloric control-diet on allergic immune responses. C57BL/6 mice weaned and maintained on control (11% fat calories) or isocaloric high-fat diet (58% fat calories) were systemically sensitized with ovalbumin and challenged in the lungs. Allergic airway inflammation was assessed by measuring lung inflammation; serum antibodies; and, cytokines in serum, bronchoalveolar lavage (BAL) fluid and in supernatants of in vitro stimulated lung draining lymph node and spleen lymphocytes. There was a significant reduction in lung eosinophilia and IL-5 in high-fat fed mice. Lung draining lymph node cells from these mice showed reduced pro-inflammatory cytokine (MCP-1 and TNF-alpha) release after ovalbumin re-stimulation and reduced release of IL-13 after concanavalin-A stimulation, indicating a general rather than just an antigen-specific change. There was no difference in IFN-gamma release. In contrast, pro-inflammatory cytokine release was increased from splenocytes. Decreased eosinophilia was not due to increased regulatory T cell or IL-10 induction in draining lymph nodes or spleen, nor to changes in antibody response to ovalbumin. However, decreased levels of serum and BAL eotaxin were found in high-fat fed animals. The data indicate that high-fat dietary content redirects local immune responses to allergen in the lungs and systemic responses in the spleen and serum. These effects are not due to changes in regulatory T cell populations but may reflect a failure to mobilize eosinophils in response to allergic challenge.

Inhaled chemicals may enhance allergic airway inflammation in ovalbumin-sensitised mice

Occupational allergy and asthma is a challenging issue in the developing countries. Chemicals inhaled in the workplaces may act not only as allergens but also as immune response modifiers, contributing to asthma exacerbation. In this study, we tested the adjuvant effect of 20 ppm chloroform, 10 ppm 1,1-dichloroethylene, and 100 ppm styrene in mice. Female BALB/c mice were sensitised to ovalbumin (OVA) without using alum. During the OVA-sensitisation period, these mice were exposed by inhalation to the chemicals studied for 6 h/day for four consecutive days. After two OVA-intratracheal challenges, a mild Th2 immune response was observed in the OVA-exposed groups. This response was characterised by a mild increase in serum specific IgE level, in local Th2 cytokine production, and in lung inflammatory reaction. Exposure to styrene or chloroform alone slightly increased Th2 cytokine production by lung-draining lymph node cells cultured with concanavaline A, except for the IL-4 level in the chloroform exposure group, which decreased. On the other hand, exposure to 1,1-dichloroethylene alone markedly increased the Th2 cytokine levels compared to those observed in the groups exposed to OVA alone. In the combined OVA + chemical-treated groups, styrene potentiated IL-4, -5 and -13 production efficiently (approximately two, four and three times higher, respectively), resulting in an increase in the total IgE levels and inflammatory reaction. On the other hand, the enhanced IgE levels and the exacerbation of the inflammatory response by 1,1-dichloroethylene or chloroform were associated with only minor changes in local cytokine levels. These findings suggest that exposure to chemicals through inhalation may aggravate the allergic lung inflammation. And this, depending on the chemical exposure conditions, may result from the synergistic effect of chemicals and allergen on local Th2 cytokine production.

Deletion of TLR3 Alters the Pulmonary Immune Environment and Mucus Production during Respiratory Syncytial Virus Infection

The detection of a viral infection by pattern recognition receptors (PAMPs) is an integral part of antiviral immunity. In these studies we have investigated the role of TLR3, which recognizes dsRNA, in Respiratory Syncytial virus (RSV) infection using B6 background mice with a TLR3 deletion. Although we observed no changes in viral growth, we did find that TLR3-/- mice demonstrated significant increases in mucus production in the airways of RSV-infected mice. The qualitative assessment was observed by examining differentially stained lungs, followed by immunohistochemical staining for gob5, a mucus-associated protein. The histopathologic observations were verified using quantitative gene expression analyses examining gob5 gene expression. Changes in pulmonary mucus production were accompanied by an increase in pulmonary IL-13 as well as IL-5 expression and eosinophils in the airways of TLR3-/- mice. Examining leukocytes in the airway indicated an accumulation of eosinophils in TLR3-/- mice, but not wild-type mice, after RSV infection. Isolated lung draining lymph node cells from TLR3-/- mice produced significant increases in Th2-type cytokines, IL-5, and IL-13, compared with wild-type TLR3+/+ mice only after RSV infection. To demonstrate a causative link, we depleted TLR3-/- mice of IL-13 during RSV infection and found that mucus and gob5 expression in the lungs was attenuated. Together, these studies highlight that although TLR3 may not be required for viral clearance, it is necessary to maintain the proper immune environment in the lung to avoid developing pathologic symptoms of disease.

Comparison of intranasal and transcutaneous immunization for induction of protective immunity against Chlamydia muridarum respiratory tract infection

Chlamydia pneumoniae causes a range of respiratory infections including bronchitis, pharyngitis and pneumonia. Infection has also been implicated in exacerbation/initiation of asthma and chronic obstructive pulmonary disease (COPD) and may play a role in atherosclerosis and Alzheimer's disease. We have used a mouse model of Chlamydia respiratory infection to determine the effectiveness of intranasal (IN) and transcutaneous immunization (TCI) to prevent Chlamydia lung infection. Female BALB/c mice were immunized with chlamydial major outer membrane protein (MOMP) mixed with cholera toxin and CpG oligodeoxynucleotide adjuvants by either the IN or TCI routes. Serum and bronchoalveolar lavage (BAL) were collected for antibody analysis. Mononuclear cells from lung-draining lymph nodes were stimulated in vitro with MOMP and cytokine mRNA production determined by real time PCR. Animals were challenged with live Chlamydia and weighed daily following challenge. At day 10 (the peak of infection) animals were sacrificed and the numbers of recoverable Chlamydia in lungs determined by real time PCR. MOMP-specific antibody-secreting cells in lung tissues were also determined at day 10 post-infection. Both IN and TCI protected animals against weight loss compared to non-immunized controls with both immunized groups gaining weight by day 10-post challenge while controls had lost 6% of body weight. Both immunization protocols induced MOMP-specific IgG in serum and BAL while only IN immunization induced MOMP-specific IgA in BAL. Both immunization routes resulted in high numbers of MOMP-specific antibody-secreting cells in lung tissues (IN > TCI). Following in vitro re-stimulation of lung-draining lymph node cells with MOMP; IFNγ mRNA increased 20-fold in cells from IN immunized animals (compared to non-immunized controls) while IFNγ levels increased 6- to 7-fold in TCI animals. Ten days post challenge non-immunized animals had >7000 IFU in their lungs, IN immunized animals <50 IFU and TCI immunized animals <1500 IFU. Thus, both intranasal and transcutaneous immunization protected mice against respiratory challenge with Chlamydia. The best protection was obtained following IN immunization and correlated with IFNγ production by mononuclear cells in lung-draining LN and MOMP-specific IgA in BAL.

LIPOPOLYSACCHARIDE-ENHANCED, TOLL-LIKE RECEPTOR 4-DEPENDENT, T HELPER CELL TYPE 2 RESPONSES TO INHALED ANTIGEN

Eisenbarth SC, Piggott DA, Huleatt JW, et al. J Exp Med. 2002;196:1645–1651To evaluate the dose-dependent effects of lipopolysaccharide (LPS) (endotoxin) inhalation and LPS-induced activation of toll-like receptor 4 (TLR4) on the generation of T helper (Th) cell type 2-dependent allergic inflammatory responses to an inhaled antigen, ovalbumin (OVA).Wild-type or TLR4-deficient, 6- to 10-week-old, female mice were studied.Mice were sensitized with intranasal exposure to LPS-depleted OVA or OVA with either a low (0.1 μg) or high (100 μg) dose of LPS. After intranasal OVA challenge, pulmonary inflammatory responses were assessed through enumerating bronchoalveolar lavage cells, performing histopathologic analyses, measuring OVA-dependent cytokine production by lung-draining lymph node cells, and determining OVA-specific serum antibody levels. Dendritic cell responses to OVA with LPS were evaluated in cytokine production, activation marker expression, and cell migration studies.After antigen challenge, mice sensitized to OVA with low-dose LPS exhibited a Th2-associated response, with pulmonary eosinophilia, airway mucus secretion, Th2 cytokine (interleukin-5 and -13) production by lymph node cells, and production of high levels of OVA-specific immunoglobulin E and immunoglobulin G1. In contrast, mice sensitized to OVA with high-dose LPS developed a Th1-associated response, with a predominance of neutrophils, no airway mucus secretion, Th1 cytokine (interferon-γ) production by lymph node cells, and production of high levels of OVA-specific immunoglobulin G2a. No pulmonary inflammatory responses were observed with mice sensitized to LPS-depleted OVA. OVA sensitization of TLR4-deficient mice with either low- or high-dose LPS failed to generate inflammatory responses. However, treatment with tumor necrosis factor, which is secreted by LPS-stimulated dendritic cells, compensated for deficient dendritic cell activation, restoring the responses of TLR4-deficient mice to OVA with either low- or high-dose LPS.The authors concluded that inhalation of low doses of LPS with OVA antigen was necessary to induce allergic or Th2-dependent responses in the lung, whereas inhalation of high doses of LPS with antigen induced nonallergic or Th1-dependent responses. The LPS-mediated effects on allergic sensitization were dependent on signaling through TLR4 on dendritic cells.The effects of endotoxin exposure on the development of atopy and asthma are seemingly paradoxical. Although studies show that exposure to endotoxin early in life inhibits the development of asthma and atopic disease, other studies demonstrate adverse effects of endotoxin on airway function. Although endotoxin is ubiquitous in children’s environments, the exposure level can vary and may be the factor influencing the development of atopy. For example, this dosage effect may help explain the protection from atopy associated with growing up on a farm, where endotoxin exposure is excessive. The results of this study are an intriguing advance toward understanding the basis for these conflicting data, suggesting a mechanism through which the dose of endotoxin present during aeroallergen exposure could influence the incidence of allergic sensitization.

Using Tolerance Induced Via the Anterior Chamber of the Eye to Inhibit Th2-Dependent Pulmonary Pathology

Anterior chamber-associated immune deviation (ACAID), a manifestation of ocular immune privilege, prevents Th1-dependent delayed hypersensitivity from developing in response to eye-derived Ags, thereby preserving vision. Since Th2-type cells have recently been shown to mediate destructive inflammation of the cornea, we wondered whether pre-emptive induction of ACAID could inhibit Th2 responses. Using a murine model of OVA -specific, Th2-dependent pulmonary inflammation, we pretreated susceptible mice by injecting OVA alone into the anterior chamber, or by injecting OVA-pulsed, TGF-beta2-treated peritoneal exudate cells i.v. These mice were then immunized with OVA plus alum strategy that generates Th2-mediated OVA-specific pulmonary pathology. When pretreated mice were challenged intratracheally with OVA, their bronchoalveolar lavage fluids contained far fewer eosinophils and significantly less IL-4, IL-5, and IL-13 compared with that of positive, nonpretreated controls. Similarly, lung-draining lymph node cells of pretreated mice secreted significantly less IL-4, IL-5, and IL-13 when challenged in vitro with OVA. Moreover, sera from pretreated mice contained much lower titers of OVA-specific IgE Abs. We conclude that Ags injected into the anterior chamber of the eye impair both Th1 and Th2 responses. These results reduce the likelihood that ACAID regulates Th1 responses via a Th2-like mechanism. Thus, immune privilege of the eye regulates inflammation secondary to both Th1- and Th2-type immune responses.