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.

In Pursuit of Optimal Outcomes: A Framework for Quality Standards in Immune Effector Cell Therapy.

Immune effector cell (IEC) therapy represents a transformative advancement in oncology, leveraging the immune system to combat various malignancies. This article outlines a comprehensive framework for establishing and maintaining quality standards in IEC therapy amidst rapid scientific and clinical advancements. We emphasize the integration of structured process measures, robust quality assurance, and meticulous outcome evaluation to ensure treatment efficacy and safety. Key components include multidisciplinary expertise, stringent accreditation protocols, and advanced data management systems, which facilitate standardized reporting and continual innovation. The collaborative effort among stakeholders-ranging from patients and healthcare providers to regulatory bodies-is crucial in delivering high-quality IEC therapies. This framework aims to enhance patient outcomes and cement the role of IEC therapy as a cornerstone of modern oncology, promoting continuous improvement and adherence to high standards across the therapeutic spectrum.

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.