Emergence, origin, and function of neutrophil-dendritic cell hybrids in experimentally induced inflammatory lesions in mice.

Although unusual neutrophils expressing major histocompatibility complex class II (MHC II) and costimulatory molecules have been detected at inflammatory sites in mice and humans, their identity, origin, and function remain unclear. We have demonstrated that, when cultured with granulocyte macrophage-colony-stimulating factor, neutrophils can give rise to a unique hybrid population exhibiting dual phenotypic and functionality of neutrophils and dendritic cells (DCs). Here we report that hybrid cells expressing surface markers of neutrophils (Ly6G, L-selectin, CXC chemokines receptor 2, and 7/4) and DCs (CD11c, MHC II, CD80, and CD86) become detectable in the peritoneal cavity, skin, lung, and lymph nodes under inflammatory conditions. Importantly, 20% to 30% of the adoptively transferred neutrophils acquired CD11c and MHC II expression when recovered from inflammatory lesions, demonstrating neutrophil → hybrid conversion in living animals. Using Escherichia coli strains expressing green fluorescent protein and ovalbumin, we further show hybrids play dual protective roles by rapidly clearing bacteria and presenting bacterial antigens to CD4 T cells. These results indicate that some of the neutrophils recruited to inflammatory lesions can differentiate into neutrophil-DC hybrids, thus challenging the classic view of neutrophils as terminally differentiated leukocytes destined to die or to participate primarily in host innate immunity.

Reduction of Baylisascaris procyonis eggs in raccoon latrines, suburban Chicago, Illinois, USA.

Baylisascaris procyonis, a common roundworm of raccoons, causes severe or fatal human infections, often in suburban areas. To evaluate the effectiveness of a baiting strategy requiring minimal labor, we distributed medicated baits near raccoon latrines in suburban Chicago, Illinois, USA. This strategy lowered B. procyonis prevalence in raccoons, possibly reducing risk to humans.

Persistence of LPS-induced lung inflammation in surfactant protein-C-deficient mice.

Pulmonary surfactant protein-C (SP-C) gene-targeted mice (Sftpc(-/-)) develop progressive lung inflammation and remodeling. We hypothesized that SP-C deficiency reduces the ability to suppress repetitive inflammatory injury. Sftpc(+/+) and Sftpc(-/-) mice given three doses of bacterial LPS developed airway and airspace inflammation, which was more intense in the Sftpc(-/-) mice at 3 and 5 days after the final dose. Compared with Sftpc(+/+)mice, inflammatory injury persisted in the lungs of Sftpc(-/-) mice 30 days after the final LPS challenge. Sftpc(-/-) mice showed LPS-induced airway goblet cell hyperplasia with increased detection of Sam pointed Ets domain and FoxA3 transcription factors. Sftpc(-/-) type II alveolar epithelial cells had increased cytokine expression after LPS exposure relative to Sftpc(+/+) cells, indicating that type II cell dysfunction contributes to inflammatory sensitivity. Microarray analyses of isolated type II cells identified a pattern of enhanced expression of inflammatory genes consistent with an intrinsic low-level inflammation resulting from SP-C deficiency. SP-C-containing clinical surfactant extract (Survanta) or SP-C/phospholipid vesicles blocked LPS signaling through the LPS receptor (Toll-like receptor [TLR] 4/CD14/MD2) in human embryonic kidney 293T cells, indicating that SP-C blocks LPS-induced cytokine production by a TLR4-dependent mechanism. Phospholipid vesicles alone did not modify the TLR4 response. In vivo deficiency of SP-C leads to inflammation, increased cytokine production by type II cells, and persistent inflammation after repetitive LPS stimulation.

Genetic replacement of surfactant protein-C reduces respiratory syncytial virus induced lung injury.

BACKGROUND: Individuals with deficiencies of pulmonary surfactant protein C (SP-C) develop interstitial lung disease (ILD) that is exacerbated by viral infections including respiratory syncytial virus (RSV). SP-C gene targeted mice (Sftpc -/-) lack SP-C, develop an ILD-like disease and are susceptible to infection with RSV. METHODS: In order to determine requirements for correction of RSV induced injury we have generated compound transgenic mice where SP-C expression can be induced on the Sftpc -/- background (SP-C/Sftpc -/-) by the administration of doxycycline (dox). The pattern of induced SP-C expression was determined by immunohistochemistry and processing by Western blot analysis. Tissue and cellular inflammation was measured following RSV infection and the RSV-induced cytokine response of isolated Sftpc +/+ and -/- type II cells determined. RESULTS: After 5 days of dox administration transgene SP-C mRNA expression was detected by RT-PCR in the lungs of two independent lines of bitransgenic SP-C/Sftpc -/- mice (lines 55.3 and 54.2). ProSP-C was expressed in the lung, and mature SP-C was detected by Western blot analysis of the lavage fluid from both lines of SP-C/Sftpc -/- mice. Induced SP-C expression was localized to alveolar type II cells by immunostaining with an antibody to proSP-C. Line 55.3 SP-C/Sftpc -/- mice were maintained on or off dox for 7 days and infected with 2.6x107 RSV pfu. On day 3 post RSV infection total inflammatory cell counts were reduced in the lavage of dox treated 55.3 SP-C/Sftpc -/- mice (p = 0.004). The percentage of neutrophils was reduced (p = 0.05). The viral titers of lung homogenates from dox treated 55.3 SP-C/Sftpc -/- mice were decreased relative to 55.3 SP-C/Sftpc -/- mice without dox (p = 0.01). The cytokine response of Sftpc -/- type II cells to RSV was increased over that of Sftpc +/+ cells. CONCLUSIONS: Transgenic restoration of SP-C reduced inflammation and improved viral clearance in the lungs of SP-C deficient mice. The loss of SP-C in alveolar type II cells compromises their response to infection. These findings show that the restoration of SP-C in Sftpc -/- mice in response to RSV infection is a useful model to determine parameters for therapeutic intervention.

Role of cockroach proteases in allergic disease.

Allergic asthma is on the rise in developed countries, and cockroach exposure is a major risk factor for the development of asthma. In recent years, a number of studies have investigated the importance of allergen-associated proteases in modulating allergic airway inflammation. Many of the studies have suggested the importance of allergen-associated proteases as having a direct role on airway epithelial cells and dendritic cells. In most cases, activation of the protease activated receptor (PAR)-2 has been implicated as a mechanism behind the potent allergenicity associated with cockroaches. In this review, we focus on recent evidence linking cockroach proteases to activation of a variety of cells important in allergic airway inflammation and the role of PAR-2 in this process. We will highlight recent data exploring the potential mechanisms involved in the biological effects of the allergen.

Reducing Baylisascaris procyonis roundworm larvae in raccoon latrines.

Baylisascaris procyonis roundworms, a parasite of raccoons, can infect humans, sometimes fatally. Parasite eggs can remain viable in raccoon latrines for years. To develop a management technique for parasite eggs, we tested anthelmintic baiting. The prevalence of eggs decreased at latrines, and larval infections decreased among intermediate hosts, indicating that baiting is effective.

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

BACKGROUND: A 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. METHODS: Mice (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. RESULTS: Exposure 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. CONCLUSIONS: Together these data suggest an important role for allergen activation of PAR-2 on mDCs in mediating Th2/Th17 cytokine production and allergic airway responses.

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.

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

BACKGROUND: Allergic 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. METHODS: Mice (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 IFNgamma levels in the lung, serum IgE levels, cellular infiltration, mucin production) and airway hyperresponsiveness were performed. RESULTS: Following 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. CONCLUSIONS: We 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.

Extracellular Hsp72, an endogenous DAMP, is released by virally infected airway epithelial cells and activates neutrophils via Toll-like receptor (TLR)-4.

BACKGROUND: Neutrophils play an important role in the pathophysiology of RSV, though RSV does not appear to directly activate neutrophils in the lower airways. Therefore locally produced cytokines or other molecules released by virally-infected airway epithelial cells are likely responsible for recruiting and activating neutrophils. Heat shock proteins (HSPs) are generally regarded as intracellular proteins acting as molecular chaperones; however, HSP72 can also be released from cells, and the implications of this release are not fully understood. METHODS: Human bronchial epithelial cells (16HBE14o-) were infected with RSV and Hsp72 levels were measured by Western blot and ELISA. Tracheal aspirates were obtained from critically ill children infected with RSV and analyzed for Hsp72 levels by ELISA. Primary human neutrophils and differentiated HL-60 cells were cultured with Hsp72 and supernatants analyzed for cytokine production. In some cases, cells were pretreated with polymyxin B prior to treatment with Hsp72. IkappaBalpha was assessed by Western blot and EMSA's were performed to determine NF-kappaB activation. HL-60 cells were pretreated with neutralizing antibody against TLR4 prior to Hsp72 treatment. Neutrophils were harvested from the bone marrow of wild type or TLR4-deficient mice prior to treatment with Hsp72. RESULTS: Infection of 16HBE14o- with RSV showed an induction of intracellular Hsp72 levels as well as extracellular release of Hsp72. Primary human neutrophils from normal donors and differentiated HL-60 cells treated with increasing concentrations of Hsp72 resulted in increased cytokine (IL-8 and TNFalpha) production. This effect was independent of the low levels of endotoxin in the Hsp72 preparation. Hsp72 mediated cytokine production via activation of NF-kappaB translocation and DNA binding. Using bone marrow-derived neutrophils from wild type and TLR4-mutant mice, we showed that Hsp72 directly activates neutrophil-derived cytokine production via the activation of TLR4. CONCLUSION: Collectively these data suggest that extracellular Hsp72 is released from virally infected airway epithelial cells resulting in the recruitment and activation of neutrophils.

Differences in susceptibility to German cockroach frass and its associated proteases in induced allergic inflammation in mice.

BACKGROUND: Cockroach exposure is a major risk factor for the development of asthma. Inhalation of fecal remnants (frass) is the likely sensitizing agent; however isolated frass has not been tested for its ability to induce experimental asthma in mice. METHODS: Mice (Balb/c or C57Bl/6) were sensitized and challenged with GC frass or GC frass devoid of proteases and measurements of airway inflammation and hyperresponsiveness were performed (interleukin (IL)-5, -13, and interferon gamma (IFNgamma) levels in bronchoalveolar lavage fluid, serum IgE levels, airway hyperresponsiveness, cellular infiltration, and mucin production). RESULTS: Sensitization and challenge of Balb/c mice with GC frass resulted in increased airway inflammation and hyperresponsiveness. C57Bl/6 mice were not susceptible to this model of sensitization; however they were sensitized to GC frass using a more aggressive sensitization and challenge protocol. In mice that were sensitized by inhalation, the active serine proteases in GC frass played a role in airway hyperresponsiveness as these mice had less airway hyperresponsiveness to acetylcholine and less mucin production. Proteases did not play a role in mediating the allergic inflammation in mice sensitized via intraperitoneal injection. CONCLUSION: While both strains of mice were able to induce experimental asthma following GC frass sensitization and challenge, the active serine proteases in GC frass only play a role in airway hyperresponsiveness in Balb/c mice that were susceptible to sensitization via inhalation. The differences in the method of sensitization suggest genetic differences between strains of mice.

CpG DNA modulates interleukin 1beta-induced interleukin-8 expression in human bronchial epithelial (16HBE14o-) cells.

BACKGROUND: Recognition of repeat unmethylated CpG motifs from bacterial DNA through Toll-like receptor (TLR-9) has been shown to induce interleukin (IL)-8 expression in immune cells. We sought to investigate the role of CpG oligodeoxynucleotides (ODN) on a human bronchial epithelial cells. METHODS: RT-PCR and Western blot analysis were used to determine expression of TLR-9 in human bronchial epithelial cells (16HBE14o-). Cells were treated with CpG ODN in the presence or absence of IL-1beta and IL-8 protein was determined using ELISA. In some cases cells were pretreated with chloroquine, an inhibitor of TLR-9 signaling, or SB202190, an inhibitor of the mitogen activated protein kinase p38, prior to treatment with IL-1beta and CpG. TLR9 siRNA was used to silence TLR9 prior to treatment with IL-1beta and CpG. IkappaBalpha and p38 were assessed by Western blot, and EMSA's were performed to determine NF-kappaB activation. To investigate IL-8 mRNA stability, cells were treated with IL-1beta in the absence or presence of CpG for 2 h and actinomycin D was added to induce transcriptional arrest. Cells were harvested at 15 min intervals and Northern blot analysis was performed. RESULTS: TLR-9 is expressed in 16HBE14o- cells. CpG synergistically increased IL-1beta-induced IL-8 protein abundance, however treatment with CpG alone had no effect. CpC (a control ODN) had no effect on IL-1beta-induced IL-8 levels. In addition, CpG synergistically upregulated TNFalpha-induced IL-8 expression. Silencing TLR9 using siRNA or pretreatment of cells with chloroquine had little effect on IL-1beta-induced IL-8 levels, but abolished CpG-induced synergy. CpG ODN had no effect on NF-kappaB translocation or DNA binding in 16HBE14o- cells. Treatment with CpG increased phosphorylation of p38 and pretreatment with the p38 inhibitor SB202190 attenuated the synergistic increase in IL-8 protein levels. Analysis of the half-life of IL-8 mRNA revealed that IL-8 mRNA had a longer half-life following the co-treatment of CpG and IL-1beta compared to treatment with IL-1beta alone. CONCLUSION: Together, these data demonstrate that CpG modulates IL-8 synthesis in the presence of a pro-inflammatory mediator utilizing TLR9 and post-transcriptional mechanisms involving the activation of p38 and stabilization of IL-8 mRNA.

Eosinophilic perifolliculitis presenting as a painful cystic ovarian mass in a woman with fibromyalgia: a case report.

BACKGROUND: Autoimmune oophoritis is characterized by an ovarian lymphocytic infiltrate and is a rare finding in women with premature ovarian failure. Eosinophilic perifolliculitis is a possible variant of autoimmune oophoritis, of which the pathogenesis and natural history are largely unknown. CASE: A 45-year-old woman, gravida 2, para 2, status post total abdominal hysterectomy, presented to her internist complaining of cyclic, throbbing, right lower quadrant pain. Her past medical history was significant forfibromyalgia. Pelvic ultrasound demonstrated a 2.3-cm, physiologic-appearing right ovarian cyst. Follow-up ultrasound showed a 2.2-cm, complex cyst on the right ovary that increased in size to 4.2 x 3.2 x 3.5 cm on repeat ultrasound 12 weeks later. Exploratory laparotomy and bilateral salpingo-oophorectomy were performed. Pathologic evaluation of the ovaries revealed a 3 x 2 cm regressing corpus luteal cyst with numerous eosinophils, lymphocytes, macrophages and plasma cells, infiltrating the cyst zoall. Serum antiovarian antibodies were positive. CONCLUSION: The patient's pathologic findings are consistent with the rare entity of eosinophilic perifolliculitis. The patient's history offibromyalgia is of particular interest given that both of these diseases may have an autoimmune etiology. Eosinophilic perifolliculitis should be considered in the differential diagnosis of premenopausal and perimenopausal women with pelvic pain and persistent cystic ovarian enlargement.

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.

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

BACKGROUND: Serine 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. METHODS: Wild 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. RESULTS: Neutrophil 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. CONCLUSIONS: Together 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.

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

BACKGROUND: Zinc 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. METHODS: Mice 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. RESULTS: Administration 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. CONCLUSIONS: This 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.

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-alpha 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-kappaB 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.

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-kappaB 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.

The role of endogenously produced extracellular hsp72 in mononuclear cell reprogramming.

Intracellular heat shock protein 72 (Hsp72) is known to serve a broad cytoprotective role. Recent data indicate that stressed cells can release Hsp72 into the extracellular compartment, although the biological function of extracellular Hsp72 remains to be fully elucidated. Because extracellular Hsp72 has been demonstrated to interact with Toll-like receptor 4, we hypothesized that endogenously produced and released Hsp72 would reprogram the mononuclear cell responses to LPS. THP-1 cells treated with LPS were used as a model for nuclear factor (NF)-kappaB activation. Heat shock conditions consisted of incubation at 43 degrees C for 1 h. Control cells were incubated at 37 degrees C. Twenty four hours after incubation, heat shock conditioned media (HSCM) and control media (CM) were centrifuged, and the respective cells were discarded. A separate group of naive THP-1 cells were then incubated with either HSCM or CM for 18 h and then stimulated with LPS (1 mug/mL). Heat shock significantly increased Hsp72 in HSCM compared with CM. In THP-1 cells transfected with an NF-kappaB luciferase reporter plasmid, the addition of HSCM attenuated subsequent LPS-mediated luciferase activity compared with cells incubated in CM. The addition of HSCM also attenuated LPS-mediated NF-kappaB-DNA binding and IkappaBalpha degradation. Heat shock protein 72-mediated inhibition of NF-kappaB activation was further corroborated by a significant decrease in TNF-alpha production. When HSCM and CM were subjected to Hsp72 depletion via adenosine triphosphate-agarose binding, LPS-mediated activation of NF-kappaB was partially restored, suggesting that Hsp72 is partially responsible for cellular reprogramming in response to HSCM. These data demonstrate that endogenously produced and released extracellular Hsp72 has the ability to reprogram the in vitro response to endotoxin in cultured human mononuclear cells.