Vladislav Dolgachev

Pulmonary IL-17E (IL-25) Production and IL-17RB+ Myeloid Cell-Derived Th2 Cytokine Production Are Dependent upon Stem Cell Factor-Induced Responses during Chronic Allergic Pulmonary Disease

In the present studies local neutralization of allergen-induced stem cell factor (SCF) leads to decreased production of Th2 cytokines, a reduction in inflammation, allergen-specific serum IgE/IgG1, and attenuation of severe asthma-like responses. The local blockade of pulmonary SCF also resulted in a significant reduction of IL-17E (IL-25). Sorted cell populations from the lung indicated that IL-25 was produced from c-kit+ cells, whereas Th2 cytokine production was primarily from c-kit− cell populations. SCF stimulated c-kit+ eosinophils produced IL-25, whereas bone marrow-derived mast cells did not. Using 4get mice that contain a IL-4-IRES-eGFP that when transcribed coexpress GFP and IL-4, our studies identified cells that comprised a CD11b+, GR1+, Ly6C+/−, c-kit−, CD4−, CD11c−, MHC class IIlow cell population as a source of IL-4 in the lung after chronic allergen challenge. In the bone marrow a similar cell was identified with approximately a third of the IL-4+ cells also expressing c-kit+. The pulmonary and bone marrow IL-4+ cell populations were significantly reduced upon local pulmonary anti-SCF treatment. Subsequently, when IL-25R was examined during the chronic allergen responses the expression was found on the IL-4+ myeloid cell population that expressed CD11b+GR1+. Interestingly, the IL-25R+ cells in the bone marrow were also all CD11b+GR1+, similar to the lung cells, but they were also all c-kit+, potentially suggesting a maturation of the bone marrow cell once it enters the lung and/or is stimulated by SCF. Overall, these studies suggest a complex relationship between SCF, bone marrow-derived IL-25-responsive myeloid cells, Th2 cytokines, and chronic allergic disease.

The sphingosine-1-phosphate receptor-1 antagonist, W146, causes early and short-lasting peripheral blood lymphopenia in mice

Agonists of the sphingosine-1-phosphate (S1P) receptors, like fingolimod (FTY720), are a novel class of immunomodulators. Administration of these compounds prevents the egress of lymphocytes from primary and secondary lymphoid organs causing peripheral blood lymphopenia. Although it is well established that lymphopenia is mediated by S1P receptor type 1 (S1P1), the exact mechanism is still controversial. The most favored hypothesis states that S1P1 agonists cause internalization and loss of the cell surface receptor on lymphocytes, preventing them to respond to S1P. Hence, S1P1 agonists would behave in vivo as functional antagonists of the receptor. For this hypothesis to be valid, a true S1P1 antagonist should also induce lymphopenia. However, it has been reported that S1P1 antagonists fail to show this effect, arguing against the concept. Our study demonstrates that a S1P1 antagonist, W146, induces a significant but transient blood lymphopenia in mice and a parallel increase in CD4+ and CD8+ lymphocytes in lymph nodes. Treatment with W146 also causes the accumulation of mature T cells in the medulla of the thymus and moreover, it induces lung edema. We show that both the S1P1 antagonist and a S1P1 agonist cause lymphopenia in vivo in spite of their different effects on receptor expression in vitro. Although the antagonist purely blocks the receptor and the agonist causes its disappearance from the cell surface, the response to the endogenous ligand is prevented in both cases. Our results support the hypothesis that lymphopenia evoked by S1P1 agonists is due to functional antagonism of S1P1 in lymphocytes.

Role of stem cell factor and bone marrow-derived fibroblasts in airway remodeling

Recent evidence suggests that bone marrow-derived fibroblasts are involved in airway remodeling in asthma, but the role and mechanism of recruitment of these fibroblasts remains unclear. Stem cell factor (SCF), a key factor in the propagation of hematopoietic stem cells, is important in the process of airway remodeling as well. To test the hypothesis that SCF is involved in the recruitment and differentiation of bone marrow-derived progenitor cells, GFP-bone marrow chimeric mice were created. These mice were then sensitized and chronically challenged with cockroach antigen to induce chronic airway disease. Fluorescence microscopy revealed an influx of significant numbers of GFP-expressing fibroblasts in the airways of these mice, which was confirmed by flow cytometric analysis of cells co-expressing both GFP and collagen I. These cells preferentially expressed c-kit, interleukin-31 receptor, and telomerase reverse transcriptase when compared with control lung-derived fibroblasts. Interestingly, SCF stimulated interleukin-31 receptor expression in bone marrow cells, whereas interleukin-31 strongly induced telomerase reverse transcriptase expression in fibroblasts. Treatment with neutralizing antibodies to SCF significantly reduced airway remodeling and suppressed the recruitment of these bone marrow-derived cells to the lung. Thus SCF in conjunction with interleukin-31 may play a significant role in airway remodeling by promoting the recruitment of bone marrow-derived fibroblast precursors into the lung with the capacity to promote lung myofibroblast differentiation.

Host susceptibility to gram-negative pneumonia after lung contusion

BACKGROUND: Lung contusion (LC) induces inflammation with high local concentrations of proinflammatory mediators stimulating chemotaxis and activation of neutrophils. LC is also a risk factor for development of pneumonia; however, the reason for this increased susceptibility is not clearly identified. We hypothesize that LC creates acute changes in the host pulmonary innate immune system that leads to vulnerability from a “second” hit bacterial infection. METHODS: Female C57Bl/6 mice underwent LC injury at time −6 hours. At 0 hours, these mice were inoculated intratracheally with 1,000 colony forming unit (CFU) of Klebsiella pneumoniae (LC+Pneu) or vehicle (LC). Control animals underwent a sham LC injury followed by pneumonia (Sham+Pneu). Bronchoalveolar lavage (BAL) fluid and lung tissue specimens were collected. Lung bacteria levels were quantified by serial dilution, plating, and counting CFUs. Cytokine levels were assayed by ELISA. Cell type identification and quantification was performed using flow cytometry. RESULTS: Survival at 72 hours was markedly different for the LC, Sham+Pneu, and LC+Pneu groups (100%, 80%, 20%, p < 0.05 Sham+Pneu vs. LC+Pneu). LC+Pneu animals had decreased pulmonary bacterial clearance at 24 hours compared with the Sham+Pneu group (4 × 107 vs. 8 × 106 CFUs, p < 0.05). BAL levels of IL-1&bgr;, IL-6, and keratinocyte chemoattractant were all significantly elevated in LC+Pneu mice compared with the Sham+Pneu group at 24 hours. Conversely, the Sham+Pneu mice had increased levels of macrophage inflammatory protein-2, total cells, macrophages, and neutrophils in BAL compared with the LC+Pneu group at 24 hours. LC+Pneu animals demonstrated changes in macrophage apoptosis and necrosis in BAL samples obtained 2 hours after induction of pneumonia when compared with the Sham+Pneu group. Both Sham+Pneu and LC+Pneu animals demonstrated an increase in the level of IL-10 in BAL fluid compared with LC animals. CONCLUSION: Acute inflammation after LC acts to modulate the presence of inflammatory cells necessary to combat gram-negative bacteria. This results in decreased bacterial clearance and increased mortality from pneumonia.

IL-17E (IL-25) and IL-17RB promote respiratory syncytial virus-induced pulmonary disease

One of the most severe pathologic responses of RSV infection is associated with overproduction of cytokines and inflammation, leading to mucus hypersecretion. This study investigated the role of IL‐25 in the development of RSV‐associated immunopathology. IL‐25 and its receptor IL‐17RB were increased following RSV infection, and IL‐25 blockade using neutralizing antibodies reduced RSV‐associated pathology, AHR, and type 2 cytokine production. Likewise, IL‐17RB−/− mice demonstrated a modified inflammatory response during RSV infection characterized by decreased Th2 and increased Th17 cytokine production. Additionally, the IL‐17RB−/− mice demonstrated significantly reduced inflammation and cytokine production in a model of RSV‐driven asthma exacerbation. These results indicate that IL‐25 regulates the inflammatory response to RSV infection and that its inhibition may enable a reduction in the severity of RSV‐associated pulmonary inflammation, including during viral‐induced asthma exacerbation.

Stem cell factor‐mediated activation pathways promote murine eosinophil CCL6 production and survival

Eosinophil activation during allergic diseases has a detrimental role in the generation of pathophysiologic responses. Stem cell factor (SCF) has recently shown an inflammatory, gene‐activating role on eosinophils and contributes to the generation of pathophysiologic changes in the airways during allergic responses. The data in the present study outline the signal transduction events that are induced by SCF in eosinophils and further demonstrate that MEK‐mediated signaling pathways are crucial for SCF‐induced CCL6 chemokine activation and eosinophil survival. SCF‐mediated eosinophil activation was demonstrated to include PI‐3K activation as well as MEK/MAPK phosphorylation pathways. Subsequent analysis of CCL6 gene activation and production induced by SCF in the presence or absence of rather specific inhibitors for certain pathways demonstrated that the MEK/MAPK pathway but not the PI‐3K pathway was crucial for the SCF‐induced CCL6 gene activation. These same signaling pathways were shown to initiate antiapoptotic events and promote eosinophil survival, including up‐regulation of BCL2 and BCL3. Altogether, SCF appears to be a potent eosinophil activation and survival factor.

Nanoemulsion Therapy for Burn Wounds Is Effective as a Topical Antimicrobial Against Gram-Negative and Gram-Positive Bacteria.

The aim of this study is to investigate the antimicrobial efficacy of two different nanoemulsion (NE) formulations against Gram-positive and Gram-negative bacteria in an in vivo rodent scald burn model. Male Sprague-Dawley rats were anesthetized and received a partial-thickness scald burn. Eight hours after burn injury, the wound was inoculated with 1 × 108 colony-forming units of Pseudomonas aeruginosa or Staphylococcus aureus. Treatment groups consisted of two different NE formulations (NB-201 and NB-402), NE vehicle, or saline. Topical application of the treatment was performed at 16 and 24 hours after burn injury. Animals were killed 32 hours after burn injury, and skin samples were obtained for quantitative wound culture and determination of dermal inflammation markers. In a separate set of experiments, burn wound progression was measured histologically after 72 hours of treatment. Both NE formulations (NB-201 and NB-402) significantly reduced burn wound infections with either P. aeruginosa or S. aureus and decreased median bacterial counts at least three logs when compared with animals with saline applications (p < .0001). NB-201 and NB-402 also decreased dermal neutrophil recruitment and sequestration into the wound as measured by myeloperoxidase (MPO) assay and histopathology (p < .05). In addition, there was a decrease in the proinflammatory dermal cytokines (interleukin 1-beta [IL-1&bgr;], IL-6, and tumor necrosis factor alpha [TNF-&agr;]) and the neutrophil chemoattractants CXCL1 and CXCL2. Using histologic examination, it was found that both NB-201 and NB-402 appeared to suppress burn wound progression 72 hours after injury. Topically applied NB-201 and NB-402 are effective in decreasing Gram-positive and Gram-negative bacteria growth in burn wounds, reducing inflammation, and abrogating burn wound progression.

Influence of lipopolysaccharide-binding protein on pulmonary inflammation in gram-negative pneumonia.

ABSTRACT Lipopolysaccharide-binding protein (LBP) is upregulated as part of the acute-phase response. Lipopolysaccharide-binding protein has a known multifunctional role in potentiating the recognition, clearance, and killing of gram-negative bacteria. In a Klebsiella pneumonia model, we previously demonstrated that LBP gene–deficient mice (LBP−/−) mice experience increased mortality when compared with wild-type (Wt) mice (98% vs. 59%). We hypothesize that LBP is essential to bacterial clearance from the lung, and its absence leads to alteration of the pulmonary inflammatory response to pneumonia. Twelve- to 16-week-old female C57Bl/6 Wt mice and age-matched LBP−/− mice were administered 1 × 103 colony-forming units of Klebsiella pneumoniae by intratracheal injection. Animals were euthanized at 6, 12, 24, or 36 h after inoculation. Lung tissue and bronchoalveolar lavage samples were obtained. Lung homogenate samples were assayed to determine quantitative bacterial load per whole lung, proinflammatory cytokine concentrations, myeloperoxidase activity, and assessment of pulmonary leukocyte populations. In vitro production of inflammatory mediators were also assayed after LPS stimulation of peritoneal macrophages isolated from Wt, Toll-like receptor 4 (TLR4)–deficient, and LBP−/− mice. The LBP−/− mice demonstrated significantly elevated levels of bacteria in the lung at 24 and 36 h when compared with Wt controls. The average lung levels of proinflammatory cytokines interleukin-1&bgr; (IL-1&bgr;), IL-6, keratinocyte-derived chemokine, and macrophage-inflammatory protein-2 were greater in the LBP−/− mice and remained elevated longer when compared with those in the Wt mice. Myeloperoxidase activity, an indicator of neutrophil content, was significantly increased at time 36 h in the LBP−/− mice. After in vitro stimulation of peritoneal macrophages with LPS, production of IL-1&bgr;, IL-6, IL-10, keratinocyte-derived chemokine, and macrophage-inflammatory protein-1&agr; were suppressed in LBP−/− and TLR4-deficient mice compared with that in Wt. Absence of a functional LBP gene results in diminished clearance of gram-negative bacteria from the pulmonary system. Failure to recognize and clear gram-negative bacteria via the LBP/TLR4 axis results in an initial delayed inflammatory response. This delay in LBP−/− mice is followed by excessive amplification and prolonged elevation of proinflammatory mediators and neutrophil sequestration within the lungs.

Toll-like receptor-9 (TLR9) is requisite for acute inflammatory response and injury following lung contusion

ABSTRACT Lung contusion (LC) is a significant risk factor for the development of acute respiratory distress syndrome. Toll-like receptor 9 (TLR9) recognizes specific unmethylated CpG motifs, which are prevalent in microbial but not vertebrate genomic DNA, leading to innate and acquired immune responses. TLR9 signaling has recently been implicated as a critical component of the inflammatory response following lung injury. The aim of the present study was to evaluate the contribution of TLR9 signaling to the acute physiologic changes following LC. Nonlethal unilateral closed-chest LC was induced in TLR9 (-/-) and wild-type (WT) mice. The mice were sacrificed at 5, 24, 48, and 72-h time points. The extent of injury was assessed by measuring bronchoalveolar lavage, cells (cytospin), albumin (permeability injury), and cytokines (inflammation). Following LC, only the TLR9 (-/-) mice showed significant reductions in the levels of albumin; release of pro-inflammatory cytokines IL-1&bgr;, IL-6, and Keratinocyte chemoattractant; production of macrophage chemoattractant protein 5; and recruitment of alveolar macrophages and neutrophil infiltration. Histological evaluation demonstrated significantly worse injury at all-time points for WT mice. Macrophages, isolated from TLR9 (-/-) mice, exhibited increased phagocytic activity at 24 h after LC compared with those isolated from WT mice. TLR9, therefore, appears to be functionally important in the development of progressive lung injury and inflammation following LC. Our findings provide a new framework for understanding the pathogenesis of lung injury and suggest blockade of TLR9 as a new therapeutic strategy for the treatment of LC-induced lung injury.

Interleukin 10 overexpression alters survival in the setting of gram-negative pneumonia following lung contusion

ABSTRACT Objective: Lung contusion injury produces a vulnerable window within the inflammatory defenses of the lung that predisposes the patient to pneumonia. Interleukin 10 (IL-10) is a known anti-inflammatory mediator produced by macrophages and capable of downregulating acute lung inflammation. We investigated the impact of increased levels of IL-10 within the lung on survival and the host response to trauma in the setting of lung contusion (LC) and gram-negative pneumonia. Design: A bitransgenic, tetracycline-inducible, lung-specific human IL-10 overexpression (IL-10 OE) mouse model and single transgenic (TG−) control mice were used. Mice underwent LC injury or sham injury (sham) at time −6 h. At time 0, animals were inoculated intratracheally with 500 colony-forming units of Klebsiella pneumoniae (pneu). Bronchoalveolar lavage fluid, lung tissue specimens, or purified macrophages were collected. Lung tissue and blood bacteria levels were quantified. Cytokine levels were assayed by enzyme-linked immunosorbent assay, and gene expression levels were evaluated by real-time polymerase chain reaction. Cell-type identification and quantification were done using real-time polymerase chain reaction and flow cytometry. Main Results: Interleukin 10 OE mice demonstrated decreased 5-day survival compared with TG− mice following LC + pneu (0 vs. 30%, P < 0.0001). Interleukin 10 OE mice had significantly higher lung bacteria counts (P = 0.02) and levels of bacteremia (P = 0.001) at 24 h. The IL-10 OE mice recruited more neutrophils into the alveoli as measured in bronchoalveolar lavage fluid compared with TG− mice. Alveolar macrophages from IL-10 OE mice displayed increased alternative activation (M2 macrophages, P = 0.046), whereas macrophages from TG− mice exhibited classic activation (M1 macrophages) and much higher intracellular bacterial killing potential (P = 0.03). Interleukin 6, keratinocyte-derived chemokine, and macrophage inflammatory protein 2 levels were significantly elevated in IL-10 OE LC + pneu animals (P < 0.05). Conclusions: Lung-specific IL-10 overexpression induces alternative activation of alveolar macrophages. This shift in macrophage phenotype decreases intracellular bacterial killing, resulting in a more pronounced bacteremia and accelerated mortality in a model of LC and pneumonia.