Female-specific enhancement of eosinophil recruitment and activation in a type 2 innate inflammation model in the lung.

A sex disparity in asthma prevalence and severity exists in humans. Multiple studies have highlighted the role of innate cells in shaping the adaptive immune system in chronic asthma. To explore the sex bias in the eosinophilic response, we delivered IL-33 to the lungs of mice and delineated the kinetics by which the inflammatory response was induced. Our data demonstrate that females recruited more eosinophils capable of responding to IL-33. Eosinophil activation occurred selectively in the lung tissue and was enhanced in females at all time points. This increase was associated with increased ex vivo type 2 cytokine and chemokine production and female-specific expansion of group 2 innate lymphoid cells lacking expression of the killer-cell lectin-like receptor G1. Our findings suggest that the enhanced eosinophilic response in females is due, firstly, to a greater proportion of eosinophils recruited to the lungs in females that can respond to IL-33; and secondly, to an enhanced production of type 2 cytokines in females. Our data provide insight into the mechanisms that guide the female-specific enhancement of eosinophil activation in the mouse and form the basis to characterize these responses in human asthmatics.

STAT6-IP-Dependent Disruption of IL-33-Mediated ILC2 Expansion and Type 2 Innate Immunity in the Murine Lung.

Recent interest has focused on innate-type cytokines as promoters of type 2 immunity and targets for drug development in asthma. IL-33 induces production of IL-4 and/or IL-13, which is associated with STAT6-dependent responses in innate cells, including group 2 innate lymphoid cells (ILC2s), macrophages, and eosinophils. Our published data show that STAT6-immunomodulatory peptide (STAT6-IP), an immunomodulatory peptide designed to inhibit the STAT6 transcription factor, reduces induction of Th2 adaptive immunity in respiratory syncytial virus infection and asthma models. Nevertheless, the mechanism of STAT6-IP-dependent inhibition has remained obscure. In this study, we demonstrate that STAT6-IP reduced IL-33-induced type 2 innate lung inflammation. Specifically, our data show that STAT6-IP reduced recruitment and activation of eosinophils as well as polarization of alternatively activated macrophages. Decreases in these cells correlated with reduced levels of IL-5 and IL-13 as well as several type 2 chemokines in the bronchoalveolar lavage fluid. STAT6-IP effectively inhibited expansion of ILC2s as well as the number of IL-5- and IL-13-producing ILC2s. Our data suggest that STAT6-IP effectively disrupts IL-13-dependent positive feedback loops, initiated by ILC2 activation, to suppress IL-33-induced type 2 innate immunity in the murine lung.

STAT6 inhibitory peptide reduces dendritic cell migration to the lymph nodes to control Th2 adaptive immunity in the mouse lung.

Type 2 immunity in the lung is promoted through the release of innate cytokines, including TSLP, from lung structural cells. These cytokines drive Type 2 immunity in part through upregulation of OX40L on dendritic cells (DCs). DCs expressing OX40L are potent inducers of Th2 differentiation. We have shown previously that STAT6 inhibitory peptide (STAT6-IP), a cell penetrating peptide designed to inhibit the STAT6 transcription factor, reduces the induction of Th2 adaptive immunity in murine models of respiratory syncytial virus infection. Here we show that intranasal administration of STAT6-IP at the time of antigen priming with ovalbumin (OVA), in conjunction with the Nod2 agonist, MDP, reduced frequencies of CD11b+ lung DCs expressing OX40L. Consistent with these reductions, fewer activated DCs were localized to the lung draining lymph nodes in STAT6-IP-treated mice. Upon OVA challenge four weeks later, mice treated with STAT6-IP at the time of OVA/MDP priming did not develop airway hyperresponsiveness (AHR) and had reduced influx of eosinophils into the airways, mucus production, and serum OVA-specific IgE levels. Our findings provide evidence that the long-lasting inhibitory effects of STAT6-IP are due in part to inhibition of DC responses that drive maladaptive Th2 adaptive immunity and allergic airways disease.

Dose-dependent effects of IL-17 on IL-13-induced airway inflammatory responses and airway hyperresponsiveness.

The Th2 cytokine IL-13 regulates several aspects of the asthmatic phenotype, including airway inflammation, airway hyperresponsiveness, and mucus production. The Th17 cytokine IL-17A is also implicated in asthma and has been shown to both positively and negatively regulate Th2-dependent responses in murine models of allergic airways disease. Our objective in this study was to better understand the role of IL-17 in airway inflammation by examining how IL-17 modifies IL-13-induced airway inflammatory responses. We treated BALB/c mice intranasally with IL-13 or IL-17 alone or in combination for 8 consecutive days, after which airway hyperresponsiveness, inflammatory cell influx into the lung, and lung chemokine/cytokine expression were assessed. As expected, IL-13 increased airway inflammation and airway hyperresponsiveness. IL-13 also increased numbers of IL-17-producing CD4(+) and γδ T cells. Treating mice with a combination of IL-13 and IL-17 reduced infiltration of IL-17(+) γδ T cells, but increased the number of infiltrating eosinophils. In contrast, coadministration of IL-13 with a higher dose of IL-17 decreased all IL-13-induced inflammatory responses, including infiltration of both IL-17(+)CD4(+) and γδ T cells. To examine the inhibitory activity of IL-17-expressing γδ T cells in this model, these cells were adoptively transferred into naive recipients. Consistent with an inhibitory role for γδ T cells, IL-13-induced infiltration of eosinophils, lymphocytes, and IL-17(+)CD4(+) T cells was diminished in recipients of the γδ T cells. Collectively, our data indicate that allergic airway inflammatory responses induced by IL-13 are modulated by both the quantity and the cellular source of IL-17.

Adjuvant-dependent regulation of interleukin-17 expressing γδ T cells and inhibition of Th2 responses in allergic airways disease.

BACKGROUND: Th2 immune responses are linked primarily to mild and moderate asthma, while Th17 cells, Interleukin-17A (IL-17) and neutrophilia have been implicated in more severe forms of disease. How Th2-dependent allergic reactions are influenced by Th17 and IL-17-γδ T cells is poorly understood. In murine models, under some conditions, IL-17 promotes Th2-biased airway inflammatory responses. However, IL-17-γδ T cells have been implicated in the inhibition and resolution of allergic airway inflammation and hyperresponsiveness (AHR). METHODS: We compared airway responses in Balb/c mice sensitized to OVA with (and without) a Th2-skewing aluminum-based adjuvant and the IL-17 skewing, complete Freund's adjuvant (CFA). AHR was measured invasively by flexiVent, while serum OVA-IgE was quantified by an enzyme immunoassay. Airway inflammatory and cytokine profiles, and cellular sources of IL-17 were assessed from bronchoalveolar lavage and/or lungs. The role of γδ T cells in these responses was addressed in OVA/CFA sensitized mice using a γδ T cell antibody. RESULTS: Following OVA challenge, all mice exhibited mixed eosinophilic/neutrophilic airway inflammatory profiles and elevated serum OVA-IgE. Whereas OVA/alum sensitized mice had moderate inflammation and AHR, OVA/CFA sensitized mice had significantly greater inflammation but lacked AHR. This correlated with a shift in IL-17 production from CD4+ to γδ T cells. Additionally, OVA/CFA sensitized mice, given a γδ TCR stimulatory antibody, showed increased frequencies of IL-17-γδ T cells and diminished airway reactivity and eosinophilia. CONCLUSIONS: Thus, the conditions of antigen sensitization influence the profile of cells that produce IL-17, the balance of which may then modulate the airway inflammatory responses, including AHR. The possibility for IL-17-γδ T cells to reduce AHR and robust eosinophilic inflammation provides evidence that therapeutic approaches focused on stimulating and increasing airway IL-17-γδ T cells may be an effective alternative in treating steroid resistant, severe asthma.

Sex Differences in IL-33-Induced STAT6-Dependent Type 2 Airway Inflammation.

Sex differences in asthma prevalence are well-documented but poorly understood. Murine models have contributed to our understanding of mechanisms that could regulate this sex disparity, though the majority of these studies have examined responses present after Th2 adaptive immunity is established. We have now investigated how sex influences acute activation of innate cell populations in the lung upon initial exposure to the model antigen, ovalbumin (OVA), in the presence of IL-33 (OVA+IL-33), to prime the lungs for type 2 immunity. We also examined how inflammatory responses induced by OVA+IL-33 were altered in mice lacking the STAT6 transcription factor, which is activated by IL-13, an effector cytokine of IL-33. Our data demonstrate that type 2 inflammation induced by OVA+IL-33 was more severe in female mice compared to males. Females exhibited greater cytokine and chemokine production, eosinophil influx and activation, macrophage polarization to the alternatively activated phenotype, and expansion of group 2 innate lymphoid cells (ILC2s). While increases in ILC2s and eosinophils were largely independent of STAT6 in both males and females, many other responses were STAT6-dependent only in female mice. Our findings indicate that a subset of type 2 inflammatory responses induced by OVA+IL-33 require STAT6 in both males and females and that enhanced type 2 inflammation in females, compared to males, is associated with greater IL-13 protein production. Our findings suggest blunted IL-13 production in males may protect against type 2 inflammation initiated by OVA+IL-33 delivery to the lung.

Nitration of respiratory epithelial cells by myeloperoxidase depends on extracellular nitrite.

To investigate peroxidase induced 3'-nitrotyrosine (3NT) formation, neutrophil derived myeloperoxidase (MPO) (0.025 microM) was directly administered to A549 epithelial cells with or without H(2)O(2) (150 microM). Little evidence of 3NT was found. In contrast, there was a dose dependent increase in intracellular NO (p<0.001, n=8) following MPO (0.025 microM) treatment, which was further enhanced (p<0.0003, n=8) by addition of H(2)O(2). Extracellular NO also increased after MPO (p<0.002, n=5) and with MPO and H(2)O(2) (p<0.004, n=5). Substantial 3NT formation was only detected following addition of nitrite (NO(2)(-), > or =100 microM), which induced a dose dependent increase in epithelial 3NT. In contrast, protein carbonyl formation and increased GSSG/GSH ratios were associated with MPO treatment even in the absence of NO(2)(-). Co-culture of A549 epithelial cells with polymorphonuclear leukocytes (PMN) (10(6)/ml) led to immunocytochemical detection of epithelial 3NT and induction of nitric oxide synthase (NOS2). However, in a Transwell system direct contact between PMN and A549 cells was necessary for immunodetection of 3NT but not of NOS2 consistent with a role for high local nitrite concentrations. These findings demonstrate dissociation between epithelial endogenous NO production and 3NT formation. Although MPO can influence cellular oxidative stress, particularly in the presence of H(2)O(2), 3NT formation requires the presence of high concentrations of NO(2)(-) in the milieu.

STAT6 inhibitory peptide given during RSV infection of neonatal mice reduces exacerbated airway responses upon adult reinfection.

Respiratory syncytial virus (RSV)-related hospitalization during infancy is strongly associated with the subsequent development of asthma. Early life RSV infection results in a Th2-biased immune response, which is also typical of asthma. Murine models of neonatal RSV infection have been developed to examine the possible contribution of RSV-driven Th2 responses to the development of airway hyper-responsiveness later in childhood. We have investigated the ability of a cell-penetrating STAT6 inhibitory peptide (STAT6-IP), when delivered selectively during neonatal RSV infection, to modify pathogenesis induced upon secondary RSV reinfection of adults 6 wk later. Neonatal STAT6-IP treatment inhibited the development of airway hyper-responsiveness (AHR) and significantly reduced lung eosinophilia and collagen deposition in adult mice following RSV reinfection. STAT6-IP-treated, RSV-infected neonates had reduced levels of both IL-4 and alternatively activated macrophages (AAMs) in the lungs. Our findings suggest that targeting STAT6 activity at the time of early-life RSV infection may effectively reduce the risk of subsequent asthma development.

Protein nitration in chronic sinusitis and nasal polyposis: role of eosinophils.

OBJECTIVES: To investigate the possible role of eosinophil peroxidase (EPO) in 3-nitrotyrosine (3NT) formation. STUDY DESIGN AND SETTING: Observational study employing immunocytochemistry to assess the presence of 3NT, inducible nitric oxide synthase (iNOS), eosinophils, mast cells, neutrophils, and lymphocytes in ethmoid sinus mucosal biopsies from normal controls and subjects with allergic and nonallergic chronic sinusitis and nasal polyposis. RESULTS: 3NT was more evident in biopsies from sinusitis patients (2.67 +/- 0.14, n = 21) than in healthy mucosa (0.43 +/- 0.2, n = 7, P < 0.01), but scores in atopic and nonatopic subjects were similar. Colocalization studies confirmed that 3NT was largely confined to eosinophils. No relationship was found between 3NT and other immune cells. 3NT detection was not correlated with the amount of immunostaining for iNOS. SIGNIFICANCE: Chronic sinusitis is accompanied by 3NT formation, which is largely restricted to the eosinophils, and likely driven by the action of eosinophil peroxidase, rather than by nitric oxide levels. EBM RATING: B-2.

Inhibition of experimental allergic airways disease by local application of a cell-penetrating dominant-negative STAT-6 peptide.

Allergic airways disease is initiated and perpetuated by an aberrant Th2 inflammatory response regulated in part by the cytokines IL-4 and IL-13, each of which induces activation of the STAT-6 transcription factor. Data from murine models indicate that the clinical manifestations of acute asthma are STAT-6 dependent, and thus, STAT-6 is a target for drug development in allergic airways disease. We designed a novel chimeric peptide (STAT-6 inhibitory peptide (STAT-6-IP)) comprised of a sequence predicted to bind to and inhibit STAT-6, fused to a protein transduction domain, to facilitate cellular uptake of the STAT-6-binding peptide. Our data demonstrate that the STAT-6-IP inhibited OVA-induced production of Th2 cytokines IL-4 and IL-13 in vitro. In contrast, the STAT-6-IP did not affect production of IFN-gamma, demonstrating specificity for Th2 cytokine inhibition. Following intranasal administration, the STAT-6-IP was localized to epithelial cells in the airways. Finally, in in vivo murine models of allergic rhinitis and asthma, intranasal delivery of the STAT-6-IP inhibited OVA-induced lung inflammation and mucus production as well as accumulation of eosinophils and IL-13 in bronchoalveolar lavage fluid and OVA-dependent airway hyperresponsiveness. Together these data show that local application of cell-penetrating peptide inhibitors of STAT-6 has significant potential for the treatment of allergic rhinitis and asthma.

NOS 1 is required for allergen-induced expression of NOS 2 in mice.

BACKGROUND: Although increased nitric oxide (NO) production in asthma is mediated largely by upregulation of the inducible form of nitric oxide synthase (iNOS, or NOS 2), some studies have suggested an important role for the usually constitutive neural NOS isoform (nNOS, or NOS 1). AIM: To investigate how NOS 1 may influence allergic inflammation, we used NOS 1 knockout mice and their wild-type (WT) controls. METHODS: Mice were sensitized and challenged with ovalbumin (OVA) using a protocol known to upregulate NOS 2 in the airways. RESULTS: In addition to expected increases in NOS 2 activity, OVA challenge led to increases in calcium-dependent NOS activity, which was accounted for by increased expression of NOS 1 at both mRNA (n = 5, p < 0.001) and protein levels (n = 5, p < 0.01). In NOS-1-deficient mice, OVA challenge induced less eosinophilia (n = 7, p < 0.05) and much less NO production (n = 10, p < 0.01) than in WT controls, reflecting not only the expected absence of NOS 1, but also lack of upregulation of NOS 2. This interaction appeared to be stimulus specific as NOS-1-deficient mice did upregulate NOS 2 following exposure to lipopolysaccharide (n = 5, p < 0.001). CONCLUSIONS: These findings underscore the importance of NOS 1 in allergic airway inflammation and suggest a mechanism by which NOS 1 may influence overall NO production in the airways.

Nitric oxide and collagen expression in allergic upper-airway disease.

BACKGROUND: The presence of nitric oxide (NO) in high concentrations has been described in the nasal mucosa of patients with untreated allergic rhinitis. We sought to examine the role of exogenous, as well as endogenous, NO in the production of collagen type I and type III by human nasal fibroblasts. METHODS: Primary cultured fibroblasts derived from eosinophilic nasal polyps were exposed to NO donors (500 microM of S-nitroso-N-acetyl-D,L-penicillamine (SNAP) 1000 micoM of 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA-NONOate)) and various other compounds over a 24-hour incubation period. Collagen production was evaluated qualitatively by immunocytochemistry and quantitatively by Western blot analysis. RESULTS: Maximally stimulated fibroblasts established a 2,2-fold increase in the production of type III collagen relative type 1, as compared with baseline. Oxyhemoglobin, an NO scavenger, abolished this effect. SNAP (500 microM) caused a 15.68 +/- 0.68% increase in collagen type I synthesis as compared with unstimulated controls (p < 0.05). In contrast, incubation with SNAP caused an increase in collagen type III production by a factor of 34.68 +/- 0.32% (p < 0.05). CONCLUSION: NO stimulates collagen expression in human nasal polyp-derived fibroblasts. This stimulation appeared to favor the up-regulation of collagen type III, leading to a shift in the ratio of collagen type I to type III production.