TLR7 Agonist Suppresses Group 2 Innate Lymphoid Cell-mediated Inflammation via IL-27-Producing Interstitial Macrophages.

Group 2 innate lymphoid cells (ILC2s) play an important role in the pathophysiology of asthma via the robust production of type 2 cytokines. Recent studies have demonstrated that TLR7 (Toll-like receptor 7) signaling skews toward a type 1 inflammatory response in asthma, which may lead to the development of novel treatment strategies. However, the effect of TLR7 signaling on ILC2-dependent nonallergic eosinophilic inflammation remains unclear. In this study, we investigated the effects of R848, a TLR7 agonist, in a mouse model of IL-33-induced eosinophilic airway inflammation. Intranasal administration of R848 decreased infiltration of airway eosinophils and ILC2s, mucus production in epithelial cells, and type 2 cytokine production. Flow cytometric analysis identified an increased number of interstitial macrophages (IMs) expressing a high level of TLR7 in the lung upon IL-33 stimulation. IL-33-induced IMs also expressed high levels of alternatively activated (M2)-type genes and chemokines (CCL17 and CCL24). However, R848 stimulation modified these gene expressions and elicited the production of IL-27. Coculture experiments revealed that IL-33-induced IMs directly suppressed ILC2 activation in response to R848. In addition, the inhibitory effects of R848 on ILC2-induced type 2 inflammation were defective in WSX-1-deficient mice lacking the IL-27 receptor. Taken together, these findings indicate that R848 stimulates IL-33-induced IMs to suppress ILC2-mediated type 2 airway inflammation via IL-27. These findings highlight the therapeutic potential of TLR7 agonists and/or IL-27 cascades in nonallergic asthma.

UDP-glucose and P2Y14 receptor amplify allergen-induced airway eosinophilia.

Airway eosinophilia is a hallmark of allergic asthma and is associated with mucus production, airway hyperresponsiveness, and shortness of breath. Although glucocorticoids are widely used to treat asthma, their prolonged use is associated with several side effects. Furthermore, many individuals with eosinophilic asthma are resistant to glucocorticoid treatment, and they have an unmet need for novel therapies. Here, we show that UDP-glucose (UDP-G), a nucleotide sugar, is selectively released into the airways of allergen-sensitized mice upon their subsequent challenge with that same allergen. Mice lacking P2Y14R, the receptor for UDP-G, had decreased airway eosinophilia and airway hyperresponsiveness compared with wild-type mice in a protease-mediated model of asthma. P2Y14R was dispensable for allergic sensitization and for the production of type 2 cytokines in the lung after challenge. However, UDP-G increased chemokinesis in eosinophils and enhanced their response to the eosinophil chemoattractant, CCL24. In turn, eosinophils triggered the release of UDP-G into the airway, thereby amplifying eosinophilic recruitment. This positive feedback loop was sensitive to therapeutic intervention, as a small molecule antagonist of P2Y14R inhibited airway eosinophilia. These findings thus reveal a pathway that can be therapeutically targeted to treat asthma exacerbations and glucocorticoid-resistant forms of this disease.

M2-like, dermal macrophages are maintained via IL-4/CCL24-mediated cooperative interaction with eosinophils in cutaneous leishmaniasis.

Tissue-resident macrophages (TRMs) maintain tissue homeostasis, but they can also provide a replicative niche for intracellular pathogens such as Leishmania How dermal TRMs proliferate and maintain their M2 properties even in the strong TH1 environment of the L. major infected dermis is not clear. Here, we show that, in infected mice lacking IL-4/13 from eosinophils, dermal TRMs shifted to a proinflammatory state, their numbers declined, and disease was attenuated. Intravital microscopy revealed a rapid infiltration of eosinophils followed by their tight interaction with dermal TRMs. IL-4-stimulated dermal TRMs, in concert with IL-10, produced a large amount of CCL24, which functioned to amplify eosinophil influx and their interaction with dermal TRMs. An intraperitoneal helminth infection model also demonstrated a requirement for eosinophil-derived IL-4 to maintain tissue macrophages through a CCL24-mediated amplification loop. CCL24 secretion was confined to resident macrophages in other tissues, implicating eosinophil-TRM cooperative interactions in diverse inflammatory settings.

Eosinophils, basophils and type 2 immune microenvironments in COPD-affected lung tissue.

Although elevated blood or sputum eosinophils are present in many patients with COPD, uncertainties remain regarding the anatomical distribution pattern of lung-infiltrating eosinophils. Basophils have remained virtually unexplored in COPD. This study mapped tissue-infiltrating eosinophils, basophils and eosinophil-promoting immune mechanisms in COPD-affected lungs.Surgical lung tissue and biopsies from major anatomical compartments were obtained from COPD patients with severity grades Global Initiative for Chronic Obstructive Lung Disease stages I-IV; never-smokers/smokers served as controls. Automated immunohistochemistry and in situ hybridisation identified immune cells, the type 2 immunity marker GATA3 and eotaxins (CCL11, CCL24).Eosinophils and basophils were present in all anatomical compartments of COPD-affected lungs and increased significantly in very severe COPD. The eosinophilia was strikingly patchy, and focal eosinophil-rich microenvironments were spatially linked with GATA3+ cells, including type 2 helper T-cell lymphocytes and type 2 innate lymphoid cells. A similarly localised and interleukin-33/ST2-dependent eosinophilia was demonstrated in influenza-infected mice. Both mice and patients displayed spatially confined eotaxin signatures with CCL11+ fibroblasts and CCL24+ macrophages.In addition to identifying tissue basophilia as a novel feature of advanced COPD, the identification of spatially confined eosinophil-rich type 2 microenvironments represents a novel type of heterogeneity in the immunopathology of COPD that is likely to have implications for personalised treatment.

Eosinophilic recruitment in thermally injured older animals is associated with worse outcomes and higher conversion to full thickness burn.

BACKGROUND: Partial burn injury in older patients is associated with higher rates of morbidity, mortality, and conversion to full thickness burn (Finnerty et al., 2009; Pham et al., 2009). Both human and mouse models demonstrate an altered systemic immune response in older subjects, however less is known about the localized response (Jeschke et al., 2016; Farinas et al., 2018; Mohs et al., 2017). We hypothesized that a mouse model could demonstrate differences in the localized inflammatory response of the old. METHODS: Six old (66 weeks) and young (8 weeks) mice received partial thickness thermal burns. Localized and systemic expression of nine chemokines (TNFalpha, MCP-1, MIP-2, S100A9, EGF, IL-10, RANTES, G-CSF, and EOTAXIN) were evaluated at day 3 after burn using Luminex analysis. Vimentin immunostaining was used to evaluate injury depth. RESULTS: Vimentin staining demonstrated increased burn depth in old mice (449±38µm) as compared to young (166±18µm) (p<0.05). Both groups exhibited increased localized expression of EOTAXIN after burn (p<0.05), however expression in old mice (83.6±6.1pg/ml) was lower than that of young (126.8±18.7pg/ml) (p<0.05). Systemically, however, old mice had increased baseline EOTAXIN expression (1332.40±110.78pg/ml) compared to young (666.12±45.8pg/ml) (p<0.005). CONCLUSIONS: EOTAXIN is one of the primary chemoattractants for selective eosinophilic recruitment and activation. While eosinophils are important for wound healing, a hyperactive eosinophilic response can result in tissue damage. We hypothesize that the increased baseline serum EOTAXIN in the old may prime their hyperactive response, and may contribute to their worse clinical outcomes. Long-term eosinophil activation requires further study, however our findings indicate a role for EOTAXIN and eosinophils in burn response.

Bone marrow type 2 innate lymphoid cells: a local source of interleukin-5 in interleukin-33-driven eosinophilia.

T helper type 2 (Th2) cells, type 2 innate lymphoid cells (ILC2s) and eosinophil progenitors have previously been described to produce interleukin-5 (IL-5) in the airways upon allergen provocation or by direct administration of IL-33. Eosinophilic airway inflammation is known to be associated with IL-5-dependent eosinophil development in the bone marrow, however, the source of IL-5 remains unclear. T helper cells, ILC2s and CD34+ progenitors have been proposed to be involved in this process, therefore, we investigated whether these cells are taking part in eosinophilopoiesis by producing IL-5 locally in the bone marrow in IL-33-driven inflammation. Airway exposure with IL-33 led to eosinophil infiltration in airways and elevated eotaxin-2/CCL24. Importantly, IL-5 production as well as expression of the IL-33 receptor increased in ILC2s in the bone marrow under this treatment. A small but significant induction of IL-5 was also found in CD34+ progenitors but not in T helper cells. Similar results were obtained by in vitro stimulation with IL-33 where ILC2s rapidly produced large amounts of IL-5, which coincided with the induction of eosinophil hematopoiesis. IL-33-mediated eosinophil production was indeed dependent on IL-5 as both airway and bone marrow eosinophils decreased in mice treated with anti-IL-5 in combination with IL-33. Interestingly, the responsiveness of ILC2s to IL-33 as well as IL-33-induced eotaxin-2/CCL24 were independent of the levels of IL-5. In summary, we demonstrate for the first time that IL-33 acts directly on bone marrow ILC2s, making them an early source of IL-5 and part of a process that is central in IL-33-driven eosinophilia.

An emerging role for eotaxins in neurodegenerative disease.

Eotaxins are C-C motif chemokines first identified as potent eosinophil chemoattractants. They facilitate eosinophil recruitment to sites of inflammation in response to parasitic infections as well as allergic and autoimmune diseases such as asthma, atopic dermatitis, and inflammatory bowel disease. The eotaxin family currently includes three members: eotaxin-1 (CCL11), eotaxin-2 (CCL24), and eotaxin-3 (CCL26). Despite having only ~30% sequence homology to one another, each was identified based on its ability to bind the chemokine receptor, CCR3. Beyond their role in innate immunity, recent studies have shown that CCL11 and related molecules may directly contribute to degenerative processes in the central nervous system (CNS). CCL11 levels increase in the plasma and cerebrospinal fluid of both mice and humans as part of normal aging. In mice, these increases are associated with declining neurogenesis and impaired cognition and memory. In humans, elevated plasma levels of CCL11 have been observed in Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, and secondary progressive multiple sclerosis when compared to age-matched, healthy controls. Since CCL11 is capable of crossing the blood-brain barrier of normal mice, it is plausible that eotaxins generated in the periphery may exert physiological and pathological actions in the CNS. Here, we briefly review known functions of eotaxin family members during innate immunity, and then focus on whether and how these molecules might participate in the progression of neurodegenerative diseases.

TPL-2 restricts Ccl24-dependent immunity to Heligmosomoides polygyrus.

TPL-2 (COT, MAP3K8) kinase activates the MEK1/2-ERK1/2 MAPK signaling pathway in innate immune responses following TLR, TNFR1 and IL-1R stimulation. TPL-2 contributes to type-1/Th17-mediated autoimmunity and control of intracellular pathogens. We recently demonstrated TPL-2 reduces severe airway allergy to house dust mite by negatively regulating type-2 responses. In the present study, we found that TPL-2 deficiency resulted in resistance to Heligmosomoides polygyrus infection, with accelerated worm expulsion, reduced fecal egg burden and reduced worm fitness. Using co-housing experiments, we found resistance to infection in TPL-2 deficient mice (Map3k8-/-) was independent of microbiota alterations in H. polygyrus infected WT and Map3k8-/-mice. Additionally, our data demonstrated immunity to H. polygyrus infection in TPL-2 deficient mice was not due to dysregulated type-2 immune responses. Genome-wide analysis of intestinal tissue from infected TPL-2-deficient mice identified elevated expression of genes involved in chemotaxis and homing of leukocytes and cells, including Ccl24 and alternatively activated genes. Indeed, Map3k8-/-mice had a significant influx of eosinophils, neutrophils, monocytes and Il4GFP+ T cells. Conditional knockout experiments demonstrated that specific deletion of TPL-2 in CD11c+ cells, but not Villin+ epithelial cells, LysM+ myeloid cells or CD4+ T cells, led to accelerated resistance to H. polygyrus. In line with a central role of CD11c+ cells, CD11c+ CD11b+ cells isolated from TPL-2-deficient mice had elevated Ccl24. Finally, Ccl24 neutralization in TPL-2 deficient mice significantly decreased the expression of Arg1, Retnla, Chil3 and Ear11 correlating with a loss of resistance to H. polygyrus. These observations suggest that TPL-2-regulated Ccl24 in CD11c+CD11b+ cells prevents accelerated type-2 mediated immunity to H. polygyrus. Collectively, this study identifies a previously unappreciated role for TPL-2 controlling immune responses to H. polygyrus infection by restricting Ccl24 production.

Eosinophil transendothelial migration induced by the bronchoalveolar lavage fluid of acute eosinophilic pneumonia.

BACKGROUND AND OBJECTIVE: Acute eosinophilic pneumonia (AEP) is characterized by a massive pulmonary infiltration of eosinophils. Mechanisms regulating the selective accumulation of eosinophils in AEP have not been fully established. The objective of this study was to evaluate the mechanisms of eosinophil accumulation in alveolar spaces through examination of bronchoalveolar lavage fluid (BALF) from AEP patients (AEP-BALF). METHODS: Eosinophils were isolated from the blood of healthy subjects and were placed on a human pulmonary microvascular endothelial cell monolayer cultured on Transwell filters (Coster, Cambridge, MA, USA). A saline control solution or BALF from patients with AEP, sarcoidosis or hypersensitivity pneumonitis was applied to the lower compartment, and the transendothelial migration of the eosinophils was evaluated. The concentrations of cytokines and chemokines in BALF were also measured. RESULTS: Transmigration of eosinophils across endothelial cells was only induced by the AEP-BALF. This transmigration was blocked by anti-ß2 integrin mAb. The concentrations of eotaxin-2 and monocyte chemotactic protein (MCP)-4, which are CC chemokine receptor (CCR) 3 ligands, were elevated in the AEP-BALF, and anti-CCR3 mAb or anti-MCP-4 mAb inhibited the AEP-BALF-induced transmigration of eosinophils. Furthermore, the concentration of leukotriene (LT) B4 was increased in the AEP-BALF, and an LTB4 receptor antagonist partially suppressed the AEP-BALF-induced transmigration of eosinophils. CONCLUSION: These findings suggest that CCR3 ligands including eotaxin-2 and MCP-4, and LTB4 play a role in the accumulation of eosinophils in AEP.

The inhibitory effect of soybean and soybean isoflavone diets on 2,4-dinitrofluorobenzene-induced contact hypersensitivity in mice.

Murine contact hypersensitivity (CHS) is one of the most frequently used animal models of human allergic contact dermatitis. We investigated the inhibitory effects of soybean and soy isoflavone (SI) diets on 2,4-dinitrofluorobenzene- (DNFB) induced CHS in mice. The DNFB-induced ear swelling was inhibited in the soy- and SI-treated groups. Histopathological investigations revealed that oral feeding of soybean and SI attenuated ear tissue edema and reduced the number of Gr-1(+) cell infiltrations into ear tissues. DNA microarray analysis showed that the expression of Ccl24, Xcl1, Ifng, and Ccl17 in the ear tissues was lower in the soy-treated mice than in the positive controls. In addition, CCL24 mRNA and protein expression in the ear tissues were more highly suppressed in the soy- and SI-treated groups. These results suggest that soybean and SI consumption downregulated the gene and protein expression of CCL24, thereby affording protection against CHS in mice.

A CCL24-dependent pathway augments eosinophilic airway inflammation in house dust mite-challenged Cd163(-/-) mice.

CD163 is a macrophage scavenger receptor with anti-inflammatory and pro-inflammatory functions. Here, we report that alveolar macrophages (AMΦs) from asthmatic subjects had reduced cell-surface expression of CD163, which suggested that CD163 might modulate the pathogenesis of asthma. Consistent with this, house dust mite (HDM)-challenged Cd163(-/-) mice displayed increases in airway eosinophils and mucous cell metaplasia (MCM). The increased airway eosinophils and MCM in HDM-challenged Cd163(-/-) mice were mediated by augmented CCL24 production and could be reversed by administration of a neutralizing anti-CCL24 antibody. A proteomic analysis identified the calcium-dependent binding of CD163 to Dermatophagoides pteronyssinus peptidase 1 (Der p1). Der p1-challenged Cd163(-/-) mice had the same phenotype as HDM-challenged Cd163(-/-) mice with increases in airway eosinophils, MCM and CCL24 production, while Der p1 induced CCL24 secretion by bone marrow-derived macrophages (BMMΦs) from Cd163(-/-) mice, but not BMMΦs from wild-type (WT) mice. Finally, airway eosinophils and bronchoalveolar lavage fluid CCL24 levels were increased in Der p1-challenged WT mice that received adoptively transferred AMΦ's from Cd163(-/-) mice. Thus, we have identified CD163 as a macrophage receptor that binds Der p1. Furthermore, we have shown that HDM-challenged Cd163(-/-) mice have increased eosinophilic airway inflammation and MCM that are mediated by a CCL24-dependent mechanism.

Nasal fluid release of eotaxin-3 and eotaxin-2 in persistent sinonasal eosinophilic inflammation.

BACKGROUND: The aim of the present study was to measure eotaxin-3 (CCL26) and eotaxin-2 (CCL24) in nasal lavage fluid of patients with different forms of chronic sinonasal eosinophilic inflammation to evaluate their role in the pathophysiology of nasal hypereosinophilia. METHODS: The study was an analytic cross-section study, level of evidence 3b. Patients (n = 80) with nasal hypereosinophilia were randomly recruited and grouped in the following categories: persistent allergic rhinitis (AR) (n = 25), nonallergic rhinitis with eosinophilia syndrome (NARES) (n = 30), and chronic rhinosinusitis with polyps (CRSwNP) (n = 25). Non-rhinitic volunteers (n = 20) were recruited as controls. CCL24 and CCL26 concentrations were measured by enzyme-linked immunosorbent assay (ELISA) Quantikine Human Immunoassays (R&D Systems, Minneapolis, MN) in nasal lavage fluids. Differential cell counts were performed by microscopic cytological examination of nasal tissue scraped from the inferior turbinate. RESULTS: Mean CCL26 levels were significantly higher (p < 0.05) in AR and in NARES (132.0 pg/mL and 187.63 pg/mL, respectively) than in the control group (13.5 pg/mL); in patients with CRSwNP, CCL26 values were increased compared to controls even though the difference was not statistically significant (58.9 pg/mL vs 16.5 pg/mL). Mean CCL24 levels measured in AR, NARES, and CRSwNP were significantly increased (p < 0.05) compared to controls (96.7 pg/mL, 135.4 pg/mL, and 107.0 pg/mL, respectively, vs 32.2 pg/mL). Moreover, we observed a significant correlation between CCL24 and CCL26 levels, evaluating them intraindividually by Spearman's rank correlation test. Finally, a significant correlation was found between CCL24 and CCL26 levels and the percentage of eosinophilic infiltration of nasal mucosa. CONCLUSION: Our data suggest that CCL26 and CCL24 are likely involved in the pathogenesis of chronic nasal hypereosinophilia, with a complex cooperation and different involvement of the various members of eotaxin family. Further studies are necessary to better understand the actual physiopathologic mechanism, possible clinical relevance, and therapeutic implications.

MicroRNA-155 is essential for T(H)2-mediated allergen-induced eosinophilic inflammation in the lung.

BACKGROUND: Allergic asthma is a chronic disease of the conducting airways characterized by T(H)2 inflammation and tissue remodeling after exposure to inhaled allergens. Although the T(H)2 profile is undisputed, the underlying molecular mechanisms leading to this abnormal T(H)2 profile remain largely unclear. MicroRNAs (miRNAs) are short noncoding RNAs that are important regulators of gene expression in the immune system. However, the role of miRNAs, specifically miR-155, in the regulation of allergic airway inflammation is unexplored. OBJECTIVES: We sought to assess the contribution of miR-155 in a mouse model of allergic airway inflammation. METHODS: To investigate a role for miR-155 in the regulation of allergic inflammation in vivo, we used miR-155 knockout (KO) and wild-type (WT) mice sensitized and exposed to ovalbumin. RESULTS: miR-155 deficiency resulted in diminished eosinophilic inflammation and mucus hypersecretion in the lungs of allergen-sensitized and allergen-challenged mice compared with WT control animals. This was supported by a reduction in T(H)2 cell numbers and airway T(H)2 cytokine levels and complete abrogation of allergen-induced airway eotaxin-2/CCL24 and periostin levels in miR-155 KO mice. Intranasal instillation of eotaxin-2/CCL24 before allergen challenge partially restored airway eosinophilia in miR-155 KO mice, and adoptive transfer of CD4(+) T cells resulted in a similar degree of airway eosinophilia in miR-155 KO and WT mice. Furthermore, the transcription factor PU.1, a negative regulator of T(H)2 cytokine production, was upregulated in the airways of allergen-challenged miR-155 KO mice compared with WT mice. CONCLUSIONS: Our data provides evidence that miR-155 contributes to the regulation of allergic airway inflammation by modulating T(H)2 responses through the transcription factor PU.1.

Listeria infection inhibits IgE production in regional lymph nodes by suppressing chemotaxis of basophils to lymph nodes.

Infection with Listeria induces a dominant shift to the Th1 immune response and inhibits the Th2 response. Papain is frequently utilized in animal models of allergies. Papain administration induces chemotaxis of basophils to regional lymph nodes (LNs) and production of interleukin (IL)-4 by basophils, resulting in a Th2-dominant status and increased IgE production in LNs. In this model, production of immunoglobulin (Ig) E by LN cells is primarily controlled by IL-4 produced by basophils. Based on this model, it was postulated that Listeria monocytogenes (Lm) infection suppresses IgE production by LN cells. Therefore, the effects of Lm infection on a papain-induced mouse model of allergies were investigated. Following s.c. injection of papain, basophils transiently migrated to draining LNs because of the effects of chemokine (C-C) motif ligand (CCL) 24 and secreted IL-4, inducing a Th2 response. Lm infection blocked recruitment of basophils into the popliteal LNs by inhibiting CCL24 production. Papain-induced class switch recombination (CSR) to IgE is inhibited by Lm infection, whereas CSR to IgG1 is not affected by the same treatment. Therefore, the CSR of IgG1 to IgE is basophil-dependent, whereas the CSR of IgM to IgG1 is basophil-independent. Hence, Lm infection suppresses CSR to IgE without affecting CSR to IgG1.

Alendronate attenuates eosinophilic airway inflammation associated with suppression of Th2 cytokines, Th17 cytokines, and eotaxin-2.

Bisphosphonates (BPs) have been widely used to treat osteoporosis. They act by inhibiting farnesyl diphosphate synthase in the mevalonate pathway. This resembles the action of statins, whose immune-modulating effect has recently been highlighted. In contrast, the effect of BPs on immune responses has not been elucidated well. In this study, we examined the effect of alendronate (ALN), a nitrogen-containing BP, on allergic airway inflammation in a mouse model. BALB/c mice were sensitized twice with OVA and challenged three times with nebulized OVA to induce eosinophilic airway inflammation. ALN was administered by an intragastric tube before each inhalation. ALN strongly suppressed airway eosinophilia and Th2, as well as Th17 cytokine production in the lung. ALN also attenuated eotaxin-2 production in the lung. Immunohistochemistry demonstrated that the major cell source of eotaxin-2 was peribronchial/perivascular macrophages, and flow cytometrical studies confirmed that ALN decreased eotaxin-2 expression in these macrophages. Furthermore, ALN attenuated eotaxin-2 production from mouse pleural macrophages and human monocyte/macrophage-like THP-1 cells in vitro. These results suggest that ALN suppressed Ag-induced airway responses in the mouse model. The suppression of eotaxin-2 production from macrophages appears to be one of ALN's immunomodulatory effects, whereas the mechanism by which ALN suppressed Th2 and Th17 responses could not be fully elucidated in this study. Although a clinical study should be conducted, ALN could be a novel therapeutic option for asthma.

Cys-leukotrienes promote fibrosis in a mouse model of eosinophil-mediated respiratory inflammation.

Leukotrienes (i.e., products of the 5-lipoxygenase pathway) are thought to be contributors to lung pathologies. Moreover, eosinophils have been linked with pulmonary leukotriene activities both as potential sources of these mediators and as responding effector cells. The objective of the present study was to define the role(s) of leukotrienes in the lung pathologies accompanying eosinophil-associated chronic respiratory inflammation. A transgenic mouse model of chronic T helper (Th) 2-driven inflammation expressing IL-5 from T cells and human eotaxin-2 locally in the lung (I5/hE2) was used to define potential in vivo relationships among eosinophils, leukotrienes, and chronic Th2-polarized pulmonary inflammation. Airway levels of cys-leukotrienes and leukotriene B4 (LTB4) are both significantly elevated in I5/hE2 mice. The eosinophil-mediated airway hyperresponsiveness (AHR) characteristic of these mice was abolished in the absence of leukotrienes (i.e., 5-lipoxygenase-deficient I5/hE2). More importantly, the loss of leukotrienes led to an unexpectedly significant decrease in collagen deposition (i.e., pulmonary fibrosis) that accompanied elevated levels of IL-4/-13 and TGF-ß in the lungs of I5/hE2 mice. Further studies using mice deficient for the LTB4 receptor (BLT-1(-/-)/I5/hE2) and I5/hE2 animals administered a cys-leukotriene receptor antagonist (montelukast) demonstrated that the AHR and the enhanced pulmonary fibrosis characteristic of the I5/hE2 model were uniquely cys-leukotriene-mediated events. These data demonstrate that, similar to allergen challenge models of wild-type mice, cys-leukotrienes underlie AHR in this transgenic model of severe pulmonary Th2 inflammation. These data also suggest that an underappreciated link exists among eosinophils, cys-leukotriene-mediated events, and fibrotic remodeling associated with elevated levels of IL-4/-13 and TGF-ß.

Absence of Toll-IL-1 receptor 8/single immunoglobulin IL-1 receptor-related molecule reduces house dust mite-induced allergic airway inflammation in mice.

Allergic asthma is a chronic inflammatory disease predominately associated with the activation of CD4(+) T helper Type 2 (Th2) cells. Innate pattern recognition receptors are widely acknowledged to shape the adaptive immune response. For example, the activation of airway epithelial Toll-like receptor-4 (TLR4) is necessary for the generation of house dust mite (HDM)-specific Th2 responses and the development of asthma in mice. Here we sought to determine whether the absence of Toll-interleukin-1 receptor (TIR)-8, a negative regulator of TLR4 signaling that is highly expressed in airway epithelial cells, would exacerbate HDM-induced asthma in a murine model. We found that Th2 but not Th1 or Th17 cytokine expression was significantly reduced in the lung and draining lymph nodes in HDM-sensitized/challenged TIR8 gene-deleted mice. Mucus-producing goblet cells, HDM-specific IgG1, and airway hyperreactivity were also significantly reduced in HDM-exposed, TIR8-deficient mice. Consistent with the attenuated Th2 response, eotaxin-2/CCL24 expression and airway and peribronchial eosinophils were significantly reduced in the absence of TIR8. In contrast, IL-17A-responsive chemokines and neutrophil numbers were unaffected. Similar findings were obtained for cockroach allergen. HDM sensitization alone up-regulated the expression of IL-1F5, a putative TIR8 ligand and inducer of IL-4. Of note, innate IL-4, IL-5, IL-13, and IL-33 cytokine expression was reduced during HDM sensitization in the absence of TIR8, as was the recruitment of conventional dendritic cells and basophils to the draining lymph nodes. Our findings suggest that TIR8 enhances the development of HDM-induced innate and adaptive Th2, but not Th1 or Th17 type immunity.

Chemokines mediate ethanol-induced exacerbations of murine cockroach allergen asthma.

Asthma imposes considerable patient and economic burdens, with the most severe cases causing the greatest affliction. Identifying stimuli that worsen asthma severity is an essential step to controlling both disease morbidity and the lessening economic impact. This study provides the first mechanistic investigation into how acute ethanol exposure will increase asthma severity in a murine model of mild cockroach allergen (CRA)-induced asthma. Outbred mice were sensitized to induce mild allergic asthma, with intratracheal CRA exposures on days 0 and 14. On day 21 mice were gavaged with water or 32% ethanol, and the third allergen exposure was given 30 min post-gavage. Asthmatic responses were measured at several time-points up to 42 h after the third allergen challenge. Ethanol-gavaged mice showed increased asthma severity within 90 min post-allergen challenge, with exacerbations lasting for 24 h. Ethanol caused greater airways obstruction, including an eightfold increase in epithelial cell mucin and increased mucus plugs, resulting in a 50% reduction in bronchiole patency. Ethanol gavage also induced significant increases in airways hyperreactivity. While T helper type 1 (Th1) and Th2 cytokines were not altered by ethanol gavage, pulmonary neutrophil and eosinophil recruitment were augmented. This increase was associated with increased chemokine production. Administration 2 h prior to ethanol gavage of a neutralizing antibody cocktail to keratinocyte-derived chemokine, macrophage inflammatory protein-2, eotaxin-1 and eotaxin-2 prevented ethanol-induced eosinophil recruitment and airways hyperreactivity. These data provide evidence that acute alcohol exposure immediately prior to a mild allergen-triggered asthmatic episode will exacerbate asthma severity mediated by increased production of chemokines.

SPLUNC1 deficiency enhances airway eosinophilic inflammation in mice.

Short palate, lung and nasal epithelium clone 1 (SPLUNC1) is enriched in normal airway lining fluid, but is significantly reduced in airway epithelium exposed to a Th2 cytokine milieu. The role of SPLUNC1 in modulating airway allergic inflammation (e.g., eosinophils) remains unknown. We used SPLUNC1 knockout (KO) and littermate wild-type (C57BL/6 background) mice and recombinant SPLUNC1 protein to determine the impact of SPLUNC1 on airway allergic/eosinophilic inflammation, and to investigate the underlying mechanisms. An acute ovalbumin (OVA) sensitization and challenge protocol was used to induce murine airway allergic inflammation (e.g., eosinophils, eotaxin-2, and Th2 cytokines). Our results showed that SPLUNC1 in the bronchoalveolar lavage fluid of OVA-challenged wild-type mice was significantly reduced (P < 0.05), which was negatively correlated with levels of lung eosinophilic inflammation. Moreover, SPLUNC1 KO mice demonstrated significantly higher numbers of eosinophils in the lung after OVA challenges than did wild-type mice. Alveolar macrophages isolated from OVA-challenged SPLUNC1 KO versus wild-type mice had higher concentrations of baseline eotaxin-2 that was amplified by LPS (a known risk factor for exacerbating asthma). Human recombinant SPLUNC1 protein was applied to alveolar macrophages to study the regulation of eotaxin-2 in the context of Th2 cytokine and LPS stimulation. Recombinant SPLUNC1 protein attenuated LPS-induced eotaxin-2 production in Th2 cytokine-pretreated murine macrophages. These findings demonstrate that SPLUNC1 inhibits airway eosinophilic inflammation in allergic mice, in part by reducing eotaxin-2 production in alveolar macrophages.

Significance of para-esophageal lymph nodes in food or aeroallergen-induced iNKT cell-mediated experimental eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a recently recognized inflammatory disorder driven by food hypersensitivity; however, the specific foods and mechanisms involved are unclear. In patients with EoE, we have found that hypersensitivities to corn and peanuts are the most common. Accordingly, we sensitized and exposed mice either intranasally or intragastrically with corn or peanut extract or saline. Esophageal eosinophilia, the genes of eosinophil-directed cytokines, and allergen-induced antibodies were examined in mice challenged with corn or peanut extract or saline. A high number of esophageal lamina propria eosinophils as well as eosinophilic microabscesses, intraepithelial eosinophils, extracellular eosinophilic granules, thickened and disrupted epithelial mucosa, and mast cell hyperplasia were observed in the esophagus of peanut or corn allergen-challenged mice. Mechanistic analysis indicated that para-esophageal lymph nodes might be critical in the trafficking of eosinophils to the esophagus and in EoE association to airway eosinophilia. Furthermore, experimentation with gene-targeted mice revealed that peanut allergen-induced EoE was dependent on eotaxin and invariant natural killer T (iNKT) cells, as CD1d and eotaxin-1/2 gene-deficient mice were protected from disease induction. Thus we provide evidence that para-esophageal lymph nodes are involved in food- or aeroallergen-induced eosinophilia and patchy EoE pathogenesis, likely a mechanism dependent on eotaxins and iNKT cells.