John R. Ledford

Mucosal sensitization to German cockroach involves protease-activated receptor-2

BackgroundAllergic asthma is on the rise in developed countries. A common characteristic of allergens is that they contain intrinsic protease activity, and many have been shown to activate protease-activated receptor (PAR)-2 in vitro. The role for PAR-2 in mediating allergic airway inflammation has not been assessed using a real world allergen.MethodsMice (wild type or PAR-2-deficient) were sensitized to German cockroach (GC) feces (frass) or protease-depleted GC frass by either mucosal exposure or intraperitoneal injection and measurements of airway inflammation (IL-5, IL-13, IL-17A, and IFNγ levels in the lung, serum IgE levels, cellular infiltration, mucin production) and airway hyperresponsiveness were performed.ResultsFollowing systemic sensitization, GC frass increased airway hyperresponsiveness, Th2 cytokine release, serum IgE levels, cellular infiltration and mucin production in wild type mice. Interestingly, PAR-2-deficient mice had similar responses as wild type mice. Since these data were in direct contrast to our finding that mucosal sensitization with GC frass proteases regulated airway hyperresponsiveness and mucin production in BALB/c mice (Page et. al. 2007 Resp Res 8:91), we backcrossed the PAR-2-deficient mice into the BALB/c strain. Sensitization to GC frass could now occur via the more physiologically relevant method of intratracheal inhalation. PAR-2-deficient mice had significantly reduced airway hyperresponsiveness, Th2 and Th17 cytokine release, serum IgE levels, and cellular infiltration compared to wild type mice when sensitization to GC frass occurred through the mucosa. To confirm the importance of mucosal exposure, mice were systemically sensitized to GC frass or protease-depleted GC frass via intraperitoneal injection. We found that removal of proteases from GC frass had no effect on airway inflammation when administered systemically.ConclusionsWe showed for the first time that allergen-derived proteases in GC frass elicit allergic airway inflammation via PAR-2, but only when allergen was administered through the mucosa. Importantly, our data suggest the importance of resident airway cells in the initiation of allergic airway disease, and could make allergen-derived proteases attractive therapeutic targets.

A TLR2 Agonist in German Cockroach Frass Activates MMP-9 Release and is Protective Against Allergic Inflammation in Mice

The role of TLR2 in modulating experimentally induced asthma is not fully understood. We recently identified that German cockroach (GC) frass contains a TLR2 ligand allowing us to investigate the role of a TLR2 agonist in a complex real world allergen in mediating allergic airway inflammation. GC frass exposure significantly increased airway inflammation, airway hyperresponsiveness and serum IgE levels in wild-type mice; however the same exposure in TLR2-deficient mice resulted in greatly exaggerated serum IgE and eosinophilia but diminished airway neutrophilia, suggesting a protective role for TLR2. Since GC frass inhalation usually induces airway neutrophilia, we queried the effect of neutrophil depletion on airway responses. Inhibition of neutrophil recruitment into the airways of naive wild-type mice before intratracheal inhalation of GC frass resulted in significantly increased levels of serum IgE and eosinophilia. Neutrophils are a rich source of MMP-9, and we found that MMP-9 levels were significantly increased in the airways of mice following exposure to GC frass. Importantly the levels of MMP-9 were significantly decreased in neutrophil-depleted and TLR2-deficient mice after exposure to GC frass, suggesting that TLR2 regulated MMP-9 release from neutrophils. Functionally, MMP-9-deficient mice had more acute allergic inflammation than wild-type mice, suggesting that MMP-9 was protective against experimentally induced asthma. These data suggest that TLR2 activation of neutrophils leads to release of MMP-9 which decreases allergic responses to GC frass. This suggests a protective role for TLR2 activation and MMP-9 release in the context of experimentally induced asthma in mice.

TLR2-Mediated Activation of Neutrophils in Response to German Cockroach Frass

It is becoming increasingly clear that innate immune mediators play a role in regulating adaptive immune responses in asthma pathogenesis. Cockroach exposure is a major risk factor for the development of asthma. In this study we asked whether German cockroach (GC) feces (frass) could initiate an innate immune response. Naive BALB/c mice were challenged with a single intratracheal inhalation of GC frass. Proinflammatory cytokines were significantly increased in the bronchoalveolar lavage fluid at 3 h and were maintained at higher than baseline levels for at least 24 h. Neutrophil migration into the airways was evident as early as 3 h but was maximal between 6 and 24 h postinhalation. The early increase in cytokine expression was independent of TLR2 or TLR4. Newly infiltrated airway neutrophils were responsible for maintaining high levels of cytokines in the airways. Using neutrophils as an early marker of the innate immune response, we show that show that neutrophils isolated from the airways following GC frass inhalation express TLR2 and release cytokines. GC frass directly affected neutrophil cytokine production via TLR2, but not TLR4, as evidenced by the use of TLR-neutralizing Abs and neutrophils from TLR-deficient mice. Activation of cytokine expression occurred via GC frass-induced NF-κB translocation and DNA binding. These data show that GC frass contains a TLR2 agonist and, to our knowledge, this is the first report of an allergen directly activating cells of the innate immune system via TLR2 and suggests an important link between innate and adaptive immunity.

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

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

German Cockroach Proteases and Protease-Activated Receptor-2 Regulate Chemokine Production and Dendritic Cell Recruitment

We recently showed that serine proteases in German cockroach (GC) feces (frass) decreased experimental asthma through the activation of protease-activated receptor (PAR)-2. Since dendritic cells (DCs) play an important role in the initiation of asthma, we queried the role of GC frass proteases in modulating CCL20 (chemokine C-C motif ligand 20) and granulocyte macrophage colony-stimulating factor (GM-CSF) production, factors that regulate pulmonary DCs. A single exposure to GC frass resulted in a rapid, but transient, increase in GM-CSF and a steady increase in CCL20 in the airways of mice. Instillation of protease-depleted GC frass or instillation of GC frass in PAR-2-deficient mice significantly decreased chemokine release. A specific PAR-2-activating peptide was also sufficient to induce CCL20 production. To directly assess the role of the GC frass protease in chemokine release, we enriched the protease from GC frass and confirmed that the protease was sufficient to induce both GM-CSF and CCL20 production in vivo. Primary airway epithelial cells produced both GM-CSF and CCL20 in a protease- and PAR-2-dependent manner. Finally, we show a decreased percentage of myeloid DCs in the lung following allergen exposure in PAR-2-deficient mice compared to wild-type mice. However, there was no difference in GC frass uptake. Our data indicate that, through the activation of PAR-2, allergen-derived proteases are sufficient to induce CCL20 and GM-CSF production in the airways. This leads to increased recruitment and/or differentiation of myeloid DC populations in the lungs and likely plays an important role in the initiation of allergic airway responses.

Early immunological response to German cockroach frass exposure induces a Th2/Th17 environment.

Cockroach exposure is a major risk factor for the development of asthma; however, the early immune events induced by cockroach leading to the Th2 response are not fully understood. Exposure of naïve mice to German cockroach (GC) feces (frass) was sufficient to induce dendritic cell (DC) recruiting and activating chemokines C-C motif ligand 20, granulocyte macrophage colony-stimulating factor, granulocyte colony-stimulating factor and macrophage inflammatory protein-1α into the airways. This corresponded with an increase in myeloid DCs (mDCs) in the airways as well as increased expression of CD80 and CD86 on the mDCs. Plasmacytoid DCs in the lung were unchanged. Levels of IL-5, IL-17A and IL-6 cytokines in whole lung cultures were significantly increased 18 h following GC frass exposure demonstrating the early development of a mixed Th2/Th17 response. In addition, GC frass stimulated the production of IL-23, IL-6 and IL-12p70 from bone marrow-derived mDCs. Adoptive transfer of GC frass-pulsed mDCs induced airway reactivity, airway inflammation as well as eosinophilia and induced a strong Th2/Th17 response in the lung. MyD88-deficient bone marrow-derived mDCs did not respond to GC frass treatment, suggesting a functional Toll-like receptor pathway was important to induce the Th2/Th17 response. Together, our data show that GC frass activated the innate immune response to augment DC recruitment and activation of mDCs which promoted robust T cell-skewing cytokines and ultimately drive the development of airway inflammation.

German Cockroach Frass Proteases Modulate the Innate Immune Response via Activation of Protease-Activated Receptor-2

Allergen exposure can induce an early innate immune response; however, the mechanism by which this occurs has not been addressed. In this report, we demonstrate a role for the active serine proteases in German cockroach (GC) feces (frass) and protease-activated receptor (PAR)-2 in modulating the innate immune response. A single exposure of GC frass induced inflammatory cytokine production and cellular infiltration in the airways of mice. In comparison, exposure to protease-depleted GC frass resulted in diminution of inflammatory cytokine production and airway neutrophilia, but had no effect on macrophage infiltration. Selective activation of PAR-2 confirmed that PAR-2 was sufficient to induce airway inflammation. Exposure of GC frass to PAR-2-deficient mice led to decreased immune responses to GC frass compared to wild-type mice. Using the macrophage as an early marker of the innate immune response, we found that GC frass induced significant release of tumor necrosis factor-α from primary alveolar macrophages. This effect was dependent on the intrinsic proteases in GC frass. We confirmed GC frass-induced cytokine expression was mediated by activation of NF-ĸB and ERK in a macrophage cell line. Collectively, these data suggest a central role for GC frass protease-PAR-2 activation in regulating the innate immune response through the activation of alveolar macrophages. Understanding the potential role of protease-PAR-2 activation as a danger signal or adjuvant could yield attractive therapeutic targets.

Zinc supplementation alters airway inflammation and airway hyperresponsiveness to a common allergen

BackgroundZinc supplementation can modulate immunity through inhibition of NF-κB, a transcription factor that controls many immune response genes. Thus, we sought to examine the mechanism by which zinc supplementation tempers the response to a common allergen and determine its effect on allergic airway inflammation.MethodsMice were injected with zinc gluconate prior to German cockroach (GC) feces (frass) exposure and airway inflammation was assessed. Primary bone marrow-derived neutrophils and DMSO-differentiated HL-60 cells were used to assess the role of zinc gluconate on tumor necrosis factor (TNF)α expression. NF-κB:DNA binding and IKK activity were assessed by EMSA and in vitro kinase assay. Protein levels of A20, RIP1 and TRAF6 were assessed by Western blot analysis. Establishment of allergic airway inflammation with GC frass was followed by administration of zinc gluconate. Airway hyperresponsiveness, serum IgE levels, eosinophilia and Th2 cytokine production were assessed.ResultsAdministration of zinc gluconate prior to allergen exposure resulted in significantly decreased neutrophil infiltration and TNFα cytokine release into the airways. This correlated with decreased NF-κB activity in the whole lung. Treatment with zinc gluconate significantly decreased GC frass-mediated TNFα production from bone-marrow derived neutrophils and HL-60 cells. We confirmed zinc-mediated decreases in NF-κB:DNA binding and IKK activity in HL-60 cells. A20, a natural inhibitor of NF-κB and a zinc-fingered protein, is a potential target of zinc. Zinc treatment did not alter A20 levels in the short term, but resulted in the degradation of RIP1, an important upstream activator of IKK. TRAF6 protein levels were unaffected. To determine the application for zinc as a therapeutic for asthma, we administered zinc following the establishment of allergic airway inflammation in a murine model. Zinc supplementation decreased airway hyperresponsiveness and serum IgE levels, but had no effect on Th2 cytokine expression.ConclusionsThis report suggests that the mechanism by which zinc supplementation alters NF-κB activity is via the alteration of A20 activity. In addition, this study provides evidence that supplementation of zinc to asthmatics may alter airway reactivity and serum IgE levels, suggesting zinc supplementation as a potential treatment for asthmatics.

Combined zinc supplementation with proinsulin C-peptide treatment decreases the inflammatory response and mortality in murine polymicrobial sepsis.

ABSTRACT Zinc is a trace element vital for immune function during host response to infection. The proinsulin C-peptide has been shown to exert beneficial effects through activation of the anti-inflammatory peroxisome proliferator–activated receptor &ggr; (PPAR&ggr;) in experimental endotoxemia. Some in vitro activities of C-peptide appear dependent on the presence of zinc. We investigated the effect of zinc supplementation before onset of sepsis on the anti-inflammatory properties of C-peptide. Male C57BL/6 mice were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP). Mice received zinc gluconate (1.3 mg/kg) intraperitoneally (i.p.) for 3 days before CLP. One hour after CLP, animals received C-peptide (280 nmol/kg i.p.) or the antimicrobial agent imipenem (25 mg/kg i.p.). Cecal ligation and puncture was associated with an 11% survival rate, pulmonary leukosequestration, and liver injury. Molecular analysis in lungs of septic mice showed increased nuclear activation of the proinflammatory extracellular signal–regulated kinases 1 and 2 and nuclear factor &kgr;B, but decreased PPAR&ggr; expression, when compared with sham animals. Combination of zinc supplementation with C-peptide posttreatment significantly improved survival rate (61%) similarly to antibiotic treatment (60%), ameliorated lung architecture and liver function, reduced tissue neutrophil infiltration, and increased bacterial clearance when compared with vehicle, C-peptide, or zinc treatment alone. These beneficial effects were associated with restored lung nuclear expression of PPAR&ggr; and reduction of phosphorylated extracellular signal–regulated kinases 1 and 2 and nuclear factor &kgr;B activities in comparison to vehicle or single treatment protocols. Our data demonstrate that short-term zinc prophylaxis before the infectious insult is a requisite for the anti-inflammatory properties of C-peptide by facilitating modulation of inflammatory pathways.

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

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