TLR10: An Intriguing Toll-Like Receptor with Many Unanswered Questions.

BACKGROUND: Toll-like receptors (TLRs) are one of the first pattern recognition receptors found in the innate immune system. The TLR family has 12 members (TLR1-TLR9, TLR11-TLR13) in mice and 10 members (TLR1-TLR10) in humans, with TLR10 being the latest identified. SUMMARY: Considerable research has been performed on TLRs; however, TLR10 is known as an orphan receptor for the lack of information on its signalling, role, and ligands. Even though there are recent studies pointing towards the potential TLR10 ligands, their function and signalling pathway are yet to be determined. KEY MESSAGES: This review gives an insight into recent findings on TLR10's pro- and anti-inflammatory properties, with the goal of outlining existing results and indicating future research topics on this receptor.

The Development and Validation of Radiopharmaceuticals Targeting Bacterial Infection.

The International Atomic Energy Agency organized a technical meeting at its headquarters in Vienna, Austria, in 2022 that included 17 experts representing 12 countries, whose research spanned the development and use of radiolabeled agents for imaging infection. The meeting focused largely on bacterial pathogens. The group discussed and evaluated the advantages and disadvantages of several radiopharmaceuticals, as well as the science driving various imaging approaches. The main objective was to understand why few infection-targeted radiotracers are used in clinical practice despite the urgent need to better characterize bacterial infections. This article summarizes the resulting consensus, at least among the included scientists and countries, on the current status of radiopharmaceutical development for infection imaging. Also included are opinions and recommendations regarding current research standards in this area. This and future International Atomic Energy Agency-sponsored collaborations will advance the goal of providing the medical community with innovative, practical tools for the specific image-based diagnosis of infection.

CD34 Protein: Its expression and function in inflammation.

CD34 has spear-headed the field of basic research and clinical transplantation since the first reports of its expression on hematopoietic stem cells (HSCs). Expressed in mice, humans, rats and other species, CD34 has been used for more than 40 years as a hematopoietic stem and progenitor cell marker. It was later found that muscle satellite cells and epidermal precursors can also be identified with the aid of CD34. Despite the usefulness of CD34 as a marker of HSCs, its overall purpose in animal physiology has remained unclear. This review recaptures CD34 structure, evolutionary conservation, proposed functions, and role in lung inflammation, to describe current research findings and to provide guidance for future studies on CD34.

Adhesion Molecules in Lung Inflammation from Repeated Glyphosate Exposures.

Glyphosate is an active ingredient in herbicides. Exposure to glyphosate-based herbicides has been associated with respiratory dysfunctions in agricultural workers. The ability of inhaled glyphosate to induce lung inflammation is not well understood. Further, the role of adhesion molecules in glyphosate-induced lung inflammation has not been studied. We evaluated lung inflammatory responses from single and repeated glyphosate exposures. Male C57BL/6 mice were intranasally exposed to glyphosate (1 µg/40 µL) for 1 day or once daily for 5 days or 10 days. Lung tissue and bronchoalveolar lavage (BAL) fluid were collected and analyzed. Repeated exposure to glyphosate for 5 days and 10 days resulted in an increase in neutrophils in BAL fluid and higher eosinophil peroxidase levels in lungs, with leukocyte infiltration further confirmed through lung histology. Repetitive exposure to glyphosate increased IL-33 and Th2 cytokines IL-5 and IL-13. A single glyphosate treatment revealed expression for ICAM-1, VCAM-1, and vWF adhesion molecules in the perivascular region of lung sections; with repeated treatment (5 and 10 days), adhesion molecule expression was found in the perivascular, peribronchiolar, and alveolar regions of the lungs. Repetitive exposure to glyphosate induced cellular inflammation in which adhesion molecules may be important to the lung inflammatory process.

Modulation of low-dose ozone and LPS exposed acute mouse lung inflammation by IF1 mediated ATP hydrolysis inhibitor, BTB06584.

Ozone and bacterial lipopolysaccharide (LPS) are common air pollutants that are related to high hospital admissions due to airway hyperreactivity and increased susceptibility to infections, especially in children, older population and individuals with underlying conditions. We modeled acute lung inflammation (ALI) by exposing 6-8 week old male mice to 0.005 ppm ozone for 2 h followed by 50 µg of intranasal LPS. We compared the immunomodulatory effects of single dose pre-treatment with CD61 blocking antibody (clone 2C9.G2), ATPase inhibitor BTB06584 against propranolol as the immune-stimulant and dexamethasone as the immune-suppressant in the ALI model. Ozone and LPS exposure induced lung neutrophil and eosinophil recruitment as measured by respective peroxidase (MPO and EPX) assays, systemic leukopenia, increased levels of lung vascular neutrophil regulatory chemokines such as CXCL5, SDF-1, CXCL13 and a decrease in immune-regulatory chemokines such as BAL IL-10 and CCL27. While CD61 blocking antibody and BTB06584 produced maximum increase in BAL leukocyte counts, protein content and BAL chemokines, these treatments induced moderate increase in lung MPO and EPX content. CD61 blocking antibody induced maximal BAL cell death, a markedly punctate distribution of NK1.1, CX3CR1, CD61. BTB06584 preserved BAL cell viability with cytosolic and membrane distribution of Gr1 and CX3CR1. Propranolol attenuated BAL protein, protected against BAL cell death, induced polarized distribution of NK1.1, CX3CR1 and CD61 but presented with high lung EPX. Dexamethasone induced sparse cell membrane distribution of CX3CR1 and CD61 on BAL cells and displayed very low lung MPO and EPX levels despite highest levels of BAL chemokines. Our study unravels ATPase inhibitor IF1 as a novel drug target for lung injury.

Expression of pentraxin 3 in equine lungs and neutrophils.

Endotoxin-induced diseases cause significant mortality and morbidity in the horse, leading to enormous economic damage to the equine industry. Neutrophils play a critical role in initiating the immune response in the lung. Pattern recognition receptors (PRRs) are programmed to recognize microbial structures unique to pathogens and mount an immune response. Pentraxin 3 (PTX3) is a PRR that is produced at sites of inflammation by many cell types upon stimulation by pro-inflammatory cytokines and agonists, such as endotoxins [also known as lipopolysaccharides (LPS)]. Pentraxin 3 recognizes and binds to many pathogens, activates the complement cascade, and has a role in the clearance of apoptotic and necrotic cells. Recently, PTX3 has been reported to be localized in the specific granules in human and mouse neutrophils, but no reports exist on the in-situ localization of PTX3 in neutrophils and the lungs of horses. Therefore, the objective of this study was to localize the PTX3 protein in normal and LPS-exposed neutrophils and in normal equine lungs. Immunohistochemical data showed PTX3 staining in the bronchial epithelial cells and the vascular endothelium of normal lungs. Immunogold electron microscopy localized PTX3 in the nuclei, cytoplasm, and vesicular organelles of alveolar macrophages, endothelial cells, and pulmonary intravascular macrophages. Immunohistochemical staining for PTX3 in isolated horse neutrophils showed an altered staining pattern in neutrophils stimulated with LPS. These data suggest that neutrophils may be a mobile form of PTX3 that is readily shuttled to the site of inflammation, where it can be released to fine tune a host defense response.

Les maladies induites par les endotoxines provoquent une mortalité et une morbidité importantes chez le cheval, entraînant d'énormes dommages économiques pour l'industrie équine. Les neutrophiles jouent un rôle essentiel dans le déclenchement de la réponse immunitaire dans les poumons. Les récepteurs de reconnaissance de formes (PRR) sont programmés pour reconnaître les structures microbiennes propres aux agents pathogènes et déclencher une réponse immunitaire. La pentraxine 3 (PTX3) est un PRR qui est produit sur les sites d'inflammation par de nombreux types de cellules lors de la stimulation par des cytokines pro-inflammatoires et des agonistes, tels que les endotoxines [également appelées lipopolysaccharides (LPS)]. La pentraxine 3 reconnaît et se lie à de nombreux agents pathogènes, active la cascade du complément et joue un rôle dans la clairance des cellules apoptotiques et nécrotiques. Récemment, il a été rapporté que PTX3 était localisé dans les granules spécifiques des neutrophiles humains et de souris, mais aucun rapport n'existe sur la localisation in situ de PTX3 dans les neutrophiles et les poumons des chevaux. Par conséquent, l'objectif de cette étude était de localiser la protéine PTX3 dans les neutrophiles normaux et exposés au LPS et dans les poumons équins normaux. Les données immunohistochimiques ont montré une coloration PTX3 dans les cellules épithéliales bronchiques et l'endothélium vasculaire des poumons normaux. La microscopie électronique d'immunomarquage à l'or colloïdal a localisé PTX3 dans les noyaux, le cytoplasme et les organites vésiculaires des macrophages alvéolaires, des cellules endothéliales et des macrophages intravasculaires pulmonaires. La coloration immunohistochimique de PTX3 dans des neutrophiles de cheval isolés a montré un schéma de coloration altéré dans les neutrophiles stimulés avec du LPS. Ces données suggèrent que les neutrophiles peuvent être une forme mobile de PTX3 qui est facilement acheminée vers le site de l'inflammation, où elle peut être libérée pour affiner une réponse de défense de l'hôte.(Traduit par Docteur Serge Messier).

Deficiency of leukocyte-specific protein 1 (LSP1) alleviates asthmatic inflammation in a mouse model.

BACKGROUND: Asthma is a major cause of morbidity and mortality in humans. The mechanisms of asthma are still not fully understood. Leukocyte-specific protein-1 (LSP-1) regulates neutrophil migration during acute lung inflammation. However, its role in asthma remains unknown. METHODS: An OVA-induced mouse asthma model in LSP1-deficient (Lsp1-/-) and wild-type (WT) 129/SvJ mice were used to test the hypothesis that the absence of LSP1 would inhibit airway hyperresponsiveness and lung inflammation. RESULTS: Light and electron microscopic immunocytochemistry and Western blotting showed that, compared with normal healthy lungs, the levels of LSP1 were increased in lungs of OVA-asthmatic mice. Compared to Lsp1-/- OVA mice, WT OVA mice had higher levels of leukocytes in broncho-alveolar lavage fluid and in the lung tissues (P < 0.05). The levels of OVA-specific IgE but not IgA and IgG1 in the serum of WT OVA mice was higher than that of Lsp1-/- OVA mice (P < 0.05). Deficiency of LSP1 significantly reduced the levels of IL-4, IL-5, IL-6, IL-13, and CXCL1 (P < 0.05) but not total proteins in broncho-alveolar lavage fluid in asthmatic mice. The airway hyper-responsiveness to methacholine in Lsp1-/- OVA mice was improved compared to WT OVA mice (P < 0.05). Histology revealed more inflammation (inflammatory cells, and airway and blood vessel wall thickening) in the lungs of WT OVA mice than in those of Lsp1-/- OVA mice. Finally, immunohistology showed localization of LSP1 protein in normal and asthmatic human lungs especially associated with the vascular endothelium and neutrophils. CONCLUSION: These data show that LSP1 deficiency reduces airway hyper-responsiveness and lung inflammation, including leukocyte recruitment and cytokine expression, in a mouse model of asthma.

Regulation of TLR10 Expression and Its Role in Chemotaxis of Human Neutrophils.

Toll-like receptors are innate immune receptors that play a critical role in pathogen-associated molecular pattern recognition. TLR10 was recently identified and very limited data are available on its expression, mechanisms that regulate its expression, and its role in primary immune cells. To study the expression pattern of TLR10 in primary immune cells, we examined TLR10 protein expression in naive and Escherichia coli lipopolysaccharide (LPS)-activated human neutrophils. Human neutrophils challenged with LPS showed a decrease in total and surface TLR10 expression at 90 min. TLR10 in LPS-activated neutrophils colocalized with flotallin-1, a lipid raft marker, and EEA-1, an early endosomal marker, to suggest its endocytosis. There was increased colocalization of TLR10 with TLR4 at LPS 60 min followed by decrease at later LPS treatment times. Treatment with TLR4 neutralizing antibody decreased cytoplasmic localization of TLR10 in LPS-treated neutrophils. Reactive oxygen species (ROS) depletion and neutralization of p65 subunit of NF-κB in LPS-treated neutrophils decreased TLR10 expression. Live cell imaging of LPS-activated neutrophils showed TLR10 translocation in the leading edge and TLR10 knockdown in neutrophils reduced their fMLP-induced chemotaxis and the number of neutrophils with pseudopodia but without affecting the expression of key proteins of actin nucleation process, ARP-3 and Diap1. Taken together, our findings show that neutrophil activation alters TLR10 expression through ROS production and NF-κB regulation, and TLR10 knockdown reduced neutrophil chemotaxis.

Localization of nucleobindin2/nesfatin-1-like immunoreactivity in human lungs and neutrophils.

Nucleobindin2 (NUCB2)/nesfatin-1 expression in human plasma positively correlates with the expression of pro-inflammatory cytokines in patients with chronic obstructive pulmonary disease (COPD), implicating its potential role in neutrophilic lung inflammation. There are no data on the localization of nucleobindin2 (NUCB2)/nesfatin-1 in human lungs and inflammatory cells. We examined the localization of NUCB2/nesfatin-1-immunoreactivity in normal and inflamed human lungs obtained from COPD patients and neutrophils with light and immunoelectron microscopy. Immunohistology showed localization of NUCB2/nesfatin-1-like immunoreactivity in the bronchiolar epithelium, alveolar septa, vascular endothelium and various immune cells of normal and inflamed lungs. Further, NUCB2/nesfatin-1-like immunoreactivity accumulated within 0.5 µm of the plasma membrane in human neutrophils following 90 min of 1 ng/mL LPS stimulation. NUCB2/nesfatin-1-like immunoreactivity was also found to localize in euchromatic portions of neutrophilic nuclei at five times the mean concentration compared to heterochromatin. Finally, our results indicate that NUCB2/nesfatin-1-like immunoreactivity is predominantly cytoplasmic including that in the Golgi complex and vesicles as it localizes at two times the concentration in neutrophilic cytoplasm compared to nucleus. Our study is the first to detail the localization of NUCB2/nesfatin-1-like immunoreactivity in lungs and neutrophils, and nuclear localization of NUCB2/nesfatin-1 also implicates its potential role in transcriptional regulation.

Characterization of Adaptive-like γδ T Cells in Ugandan Infants during Primary Cytomegalovirus Infection.

Gamma-delta (γδ) T cells are unconventional T cells that help control cytomegalovirus (CMV) infection in adults. γδ T cells develop early in gestation, and a fetal public γδ T cell receptor (TCR) clonotype is detected in congenital CMV infections. However, age-dependent γδ T cell responses to primary CMV infection are not well-understood. Flow cytometry and TCR sequencing was used to comprehensively characterize γδ T cell responses to CMV infection in a cohort of 32 infants followed prospectively from birth. Peripheral blood γδ T cell frequencies increased during infancy, and were higher among CMV-infected infants relative to uninfected. Clustering analyses revealed associations between CMV infection and activation marker expression on adaptive-like Vδ1 and Vδ3, but not innate-like Vγ9Vδ2 γδ T cell subsets. Frequencies of NKG2C+CD57+ γδ T cells were temporally associated with the quantity of CMV shed in saliva by infants with primary infection. The public γδ TCR clonotype was only detected in CMV-infected infants <120 days old and at lower frequencies than previously described in fetal infections. Our findings support the notion that CMV infection drives age-dependent expansions of specific γδ T cell populations, and provide insight for novel strategies to prevent CMV transmission and disease.

Lung inflammation from repeated exposure to LPS and glyphosate.

Agricultural workplaces consist of multiple airborne contaminants and inhalation exposures induce respiratory effects in workers. Endotoxin (LPS) and glyphosate are two common airborne contaminants in agricultural environments. We have previously shown that exposure to a combination of LPS and glyphosate synergistically modulates immune reactions as compared to individual exposures. The immunopathogenesis of acute and chronic exposure to complex agricultural exposures including LPS and glyphosate is not known; therefore, we further investigated the lung cellular inflammatory differences in mice exposed to either a combination, or individual, LPS, and glyphosate for 1 day, 5 days, and 10 days. Exposure to a combination of LPS and glyphosate resulted in greater cellular inflammatory effects in lungs as compared to individual exposures to LPS or glyphosate. Repeated exposures to the combination of LPS and glyphosate resulted in robust infiltration of inflammatory cells in the perivascular, peribronchiolar, and alveolar regions, and increases of alveolar septal thicknesses and perivascular spaces in the lungs with intense intercellular adhesion molecule (ICAM) - 1 staining in the perivascular region, but minimal staining in the pulmonary artery endothelium.

CD34 protein is expressed in murine, canine, and porcine lungs.

The cell surface protein CD34 is expressed in various human tissues and cells, including hematopoietic stem cells, vascular endothelial cells, mucosal dendritic cells, mast cells, eosinophils, microglia, fibrocytes, muscle satellite cells, and platelets. There is a lack of data on the expression of CD34 in canine and porcine tissues. Therefore, we designed a series of immunoblotting, immunohistochemistry, and immunofluorescence experiments to observe CD34 expression in murine, canine, and porcine lungs. We used a rabbit antibody (clone EP373Y) to target the conserved human CD34 C-terminal region and validated its immunoreactivity against mouse lung homogenates. The data showed diffuse bronchiolar and alveolar epithelial localization of CD34 protein in normal murine, canine, and porcine lungs. At 9 or 24 h after bacterial endotoxin exposure, murine CD34 protein shifted to specific bronchoalveolar cells with a punctate pattern, as quantified by CD34 fluorescence. Specific porcine bronchoalveolar cells and leukocytes had significant CD34-positive immunostaining after H3N1 influenza infection. Thus, our study provides fundamental data on the expression of CD34 in lungs and validates an antibody for use in further experiments in these animal species.

La protéine de surface cellulaire CD34 est exprimée dans divers tissus et cellules humains, y compris les cellules souches hématopoïétiques, les cellules endothéliales vasculaires, les cellules dendritiques des muqueuses, les mastocytes, les éosinophiles, la microglie, les fibrocytes, les cellules satellites musculaires et les plaquettes. Il existe un manque de données sur l'expression de CD34 dans les tissus canins et porcins. Par conséquent, nous avons conçu une série d'expériences d'immunobuvardage, d'immunohistochimie et d'immunofluorescence pour observer l'expression de CD34 dans les poumons murins, canins et porcins. Nous avons utilisé un anticorps de lapin (clone EP373Y) pour cibler la région C-terminale conservée du CD34 humain et validé son immunoréactivité contre des homogénats pulmonaires de souris. Les données ont montré une localisation épithéliale bronchiolaire et alvéolaire diffuse de la protéine CD34 dans les poumons normaux murins, canins et porcins. À 9 ou 24 h après l'exposition à l'endotoxine bactérienne, la protéine CD34 murine s'est déplacée vers des cellules bronchoalvéolaires spécifiques avec un motif ponctué, tel que quantifié par la fluorescence envers CD34. Des cellules bronchoalvéolaires et des leucocytes porcins spécifiques présentaient une immunocoloration significativement positive pour CD34 après une infection par le virus de l'influenza H3N1. Ainsi, notre étude fournit des données fondamentales sur l'expression de CD34 dans les poumons et valide un anticorps à utiliser dans d'autres expériences chez ces espèces animales.(Traduit par Docteur Serge Messier).

Research article expression of surfactant protein-A and D, and CD9 in lungs of 1 and 30 day old foals.

BACKGROUND: Respiratory diseases are a major cause of morbidity and mortality in the horses of all ages including foals. There is limited understanding of the expression of immune molecules such as tetraspanins and surfactant proteins (SP) and the regulation of the immune responses in the lungs of the foals. Therefore, the expression of CD9, SP-A and SP-D in foal lungs was examined. RESULTS: Lungs from one day old (n = 6) and 30 days old (n = 5) foals were examined for the expression of CD9, SP-A, and SP-D with immunohistology and Western blots. Western blot data showed significant increase in the amount of CD9 protein (p = 0.0397) but not of SP-A and SP-D at 30 days of age compared to one day. Immunohistology detected CD9 in the alveolar septa and vascular endothelium but not the bronchiolar epithelium in the lungs of the foals in both age groups. SP-A and SP-D expression was localized throughout the alveolar septa including type II alveolar epithelial cells and the vascular endothelium of the lungs in all the foals. Compared to one day old foals, the expression of SP-A and SP-D appeared to be increased in the bronchiolar epithelium of 30 day old foals. Pulmonary intravascular macrophages were also positive for SP-A and SP-D in 30 days old foals and these cells are not developed in the day old foals. CONCLUSIONS: This is the first data on the expression of CD9, SP-A and SP-D in the lungs of foals.

Pulmonary inflammatory response from co-exposure to LPS and glyphosate.

Agricultural airborne work exposures are complex in nature and workplace exposures are a risk for respiratory outcomes in workers. Endotoxin and glyphosate are two common agents in agricultural exposures. While endotoxin (lipopolysaccaride, LPS) is a potent inflammatory agent it explains only a portion of the respiratory inflammatory response. The inflammatory potential when LPS is presented with another common agricultural respiratory agent, glyphosate, is not known. METHODS: Mice were assigned to four treatment groups: control, LPS alone, glyphosate alone, glyphosate and LPS combined. Treatments were for 1, 5 or 10 days. RESULTS: Five days of repeated exposure to the comintation of LPS and glyphosate resulted in higher neutrophil counts, myloperoxidase, TNF-α, IL-6, KC levels, and ICAM-1 and TLR-2 expression compared to the same length of treatment to LPS or glyphosate alone. After 10-days of exposure, inflammatory responses decreased, however leukocyte infiltration persisted along with increases in IL-4. CONCLUSIONS: Glyphosate exposure modified LPS induced lung inflammatory responses and TLR-2 may be important in the modulated inflammatory response.

Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury.

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or fungal) inflammatory conditions. An overwhelming host response may result in compromised respiration and acute lung injury, which is characterized by lung neutrophil recruitment as a result of the patho-logical host immune, coagulative and tissue remodeling response. Sensitive microscopic methods to visualize and quantify murine lung cellular adaptations, in response to low-dose (0.05 ppm) ozone, a potent environmental pollutant in combination with bacterial lipopolysaccharide, a TLR4 agonist, are crucial in order to understand the host inflammatory and repair mechanisms. We describe a comprehensive fluorescent microscopic analysis of various lung and systemic body compartments, namely the broncho-alveolar lavage fluid, lung vascular perfusate, left lung cryosections, and sternal bone marrow perfusate. We show damage of alveolar macrophages, neutrophils, lung parenchymal tissue, as well as bone marrow cells in correlation with a delayed (up to 36-72 h) immune response that is marked by discrete chemokine gradients in the analyzed compartments. In addition, we present lung extracellular matrix and cellular cytoskeletal interactions (actin, tubulin), mitochondrial and reactive oxygen species, anti-coagulative plasminogen, its anti-angiogenic peptide fragment angiostatin, the mitochondrial ATP synthase complex V subunits, α and ß. These surrogate markers, when supplemented with adequate in vitro cell-based assays and in vivo animal imaging techniques such as intravital microscopy, can provide vital information towards understanding the lung response to novel immunomodulatory agents.

Loss of Nucleobindin-2/Nesfatin-1 increases lipopolysaccharide-induced murine acute lung inflammation.

NUCB2/nesfatin-1 is expressed in variety of tissues. Treatment with nesfatin-1 reduces inflammation in rat models of subarachnoid hemorrhage-induced oxidative brain damage and traumatic brain injury as well as myocardial injury. There is only one study showing anti-inflammatory actions of nesfatin-1 on acute lung inflammation. To more precisely determine the role of NUCB2/nesfatin-1 in acute lung inflammation, we conducted a study using NUCB2/nesfatin-1 knockout (NKO) mice as well as neutrophils isolated from the bone marrows of WT and NKO mice. Our findings suggest that the absence of NUCB2/nesfatin-1 significantly increases the accumulation of adherent neutrophils by approximately 3 times compared with WT within LPS-treated lungs. Integrating this with observations from both BALF and neutrophil cytokine expression, we propose that although neutrophils lacking NUCB2/nesfatin-1 individually secrete less pro-inflammatory cytokines compared with stimulated WT cells, the result of knocking out NUCB2/nesfatin-1 is net pro-inflammatory. No change was found in NUCB2/nesfatin-1 mRNA or protein expression comparing WT LPS and PBS-treated samples. Taken together, our results show that NUCB2/nesfatin-1 is constitutively expressed in mouse lungs and neutrophils and demonstrates anti-inflammatory properties in mouse lungs during acute lung injury, by inhibiting adherent neutrophil accumulation and inflammatory cytokine expression.

Lack of CD34 delays bacterial endotoxin-induced lung inflammation.

BACKGROUND: CD34, a pan-selectin binding protein when glycosylated, has been shown to be involved in leukocyte migration to the site of inflammation. However, only one report is available on the expression and role of CD34 in neutrophil recruitment during acute lung inflammation. METHODS: We proceeded to study the role of CD34 in lung neutrophil migration using mouse model of endotoxin induced acute lung inflammation and studied over multiple time points, in generic CD34 knock-out (KO) strain. RESULTS: While there was no difference in BAL total or differential leukocyte counts, lung MPO content was lower in LPS exposed KO compared to WT group at 3 h time-point (p = 0.0308). The MPO levels in CD34 KO mice begin to rise at 9 h (p = 0.0021), as opposed to an early 3 h rise in WT mice (p = 0.0001), indicating that KO mice display delays in lung neutrophil recruitment kinetics. KO mice do not loose endotoxin induced lung vascular barrier properties as suggested by lower BAL total protein at 3 h (p = 0.0452) and 24 h (p = 0.0113) time-points. Several pro-inflammatory cytokines and chemokines (TNF-α, IL-1ß, KC, MIP-1α, IL-6, IL-10 and IL-12 p70 sub-unit; p < 0.05) had higher levels in WT compared to KO group, at 3 h. Lung immunofluorescence in healthy WT mice reveals CD34 expression in the bronchiolar epithelium, in addition to alveolar septa. CONCLUSION: Thus, given CD34's pan-selectin affinity, and expression in the bronchiolar epithelium as well as alveolar septa, our study points towards a role of CD34 in lung neutrophil recruitment but not alveolar migration, cytokine expression and lung inflammation.

Lack of CD34 produces defects in platelets, microparticles, and lung inflammation.

Lung innate immune activation results in acute lung inflammation, which is characterized by alveolar barrier disruption and accumulation of cellular lung aggregates comprising neutrophils, platelets, mononuclear cells, and microparticles. CD34 is a sialomucin, with pan-selectin affinity and recently shown to protect the endothelial barrier in a bleomycin-induced lung injury model. However, there is very little information about the fundamental role of CD34 in regulation of the lung innate immune response. We hypothesized that CD34 regulates leukocyte recruitment by promoting optimal platelet activation (aggregation and spread) during bacterial lipopolysaccharide (LPS)-induced acute lung injury. Therefore, we utilized CD34 knock-out (KO) and wild-type (WT) mice to analyze and compare the morphology and expression of leukocyte subsets from the pulmonary and systemic compartments. We utilized the chemotactic N-formylated tri-peptide, fMLP, to understand platelet aggregation in vitro, and the fundamental immune stimulant, LPS, to induce lung injury and understand platelet activation ex vivo. Our data reveal that under steady-state conditions, KO mice possess large aggregates of integrin ß3 (CD61)-positive microparticles in peripheral blood. Moreover, the KO mice recruit a large number of neutrophils to lungs, which are not cleared even at 36-h post-LPS exposure. The KO mice display an increased platelet CD61 expression, which aggregates, but does not spread normally in response to in vitro fMLP treatment. The KO platelets display similar deficits in their spreading ability even after ex vivo LPS exposure. Thus, our data demonstrate that CD34 modulates platelet biology, microparticle aggregation, and neutrophil recruitment during murine lung inflammation.

Characterization of low-dose ozone-induced murine acute lung injury.

Ozone is a toxic and highly reactive gaseous oxidizing chemical with well-documented adverse health effects in humans. On the basis of animal and human data, environmental guidelines and air quality standards recommend a threshold for exposure of no more than 0.063 ppm of ozone (daily concentrations). This research describes a standardized sensitive model of sterile murine lung inflammation induced by exposing mice to acute (0, 4 or 24 hr), yet low, levels of ozone (0.005, 0.05 or 0.5 ppm), one that are below the current recommendations for what is considered a safe or "ambient" ozone concentration for humans. Ozone led to concentration and time-dependent phlogistic cell death in the bronchoalveolar lavage, lung epithelial damage and hemorrhage. Interestingly, we observed distinct large bright CD11b positive cells in the bronchoalveolar lavage, upregulation of lung vascular and alveolar ATP synthase as well as plasminogen and bronchiolar angiostatin expression in ozone-exposed mice, platelet and neutrophil accumulation in the lung vasculature and an eotaxin-2, IL-16, CXCL5, CXCL12, and CXCL13 dominant inflammatory response leading to lung injury. Using a fluorescent intravital microscopy set up, we quantified ozone-induced extensive alveolar cellular damage. We observed ozone-induced actin filament disorganization, perturbed respiratory mechanics, acute suppression of the alveolar reactive oxygen species (ROS) production and mitochondrial potential in ventilated lungs. We present evidence of systemic, as well as pulmonary toxicity, at 40-fold lower ozone concentrations than previously reported in mice. The findings are important in establishing a sensitive means of quantifying structural and functional lung disorganization following exposure to an aerosolized pollutant, even at levels of ozone exposure previously thought to be safe in humans.