Mast cells protect against Pseudomonas aeruginosa-induced lung injury.

Pseudomonas aeruginosa, an opportunistic pathogen, is the leading cause of morbidity and mortality in immune-compromised individuals. Maintaining the integrity of the respiratory epithelium is critical for an effective host response to P. aeruginosa. Given the close spatial relationship between mast cells and the respiratory epithelium, and the importance of tightly regulated epithelial permeability during lung infections, we examined whether mast cells influence airway epithelial integrity during P. aeruginosa lung infection in a mouse model. We found that mast cell-deficient Kit(W-sh)/Kit(W-sh) mice displayed greatly increased epithelial permeability, bacterial dissemination, and neutrophil accumulation compared with wild-type animals after P. aeruginosa infection; these defects were corrected on reconstitution with mast cells. An in vitro Transwell co-culture model further demonstrated that a secreted mast cell factor decreased epithelial cell apoptosis and tumor necrosis factor production after P. aeruginosa infection. Together, our data demonstrate a previously unrecognized role for mast cells in the maintenance of epithelial integrity during P. aeruginosa infection, through a mechanism that likely involves prevention of epithelial apoptosis and tumor necrosis factor production. Our understanding of mechanisms of the host response to P. aeruginosa will open new avenues for the development of successful preventative and treatment strategies.

Neutrophil transintestinal epithelial migration to CXCR2 ligands is regulated by adenosine.

OBJECTIVES: Polymorphonuclear leukocytes (PMN) feature prominently in the mucosa, including in crypt abscesses, of patients with inflammatory bowel disease, yet the mediators that are responsible for this migration are unknown. We discovered that CXCR2 chemokines (reportedly elevated in the mucosa) have reduced potency recruiting PMN across epithelial cell monolayers versus acellular filters, so the objective was to determine what molecules modify transepithelial PMN migration to CXCR2 chemokines. METHODS: Transwells with T84 colon carcinoma monolayers or no epithelium were used with adolescent patient peripheral blood PMN and CXCL8 (interleukin-8 [IL-8], binds CXCR1 and CXCR2), CXCL5 (epithelial-derived neutrophil chemoattractant-78 [ENA-78]), or CXCL1 (Gro-α, both bind CXCR2) as chemoattractants. RESULTS: IL-8 was equally potent at recruiting PMN across filters and T84 monolayers growing on the filters. In contrast, ENA-78 and Gro-α were significantly less potent at recruiting PMN across monolayers than across bare filters. Blocking CXCR1 reduced PMN migration across monolayers to IL-8. We ruled out superoxide radicals possibly enhancing migration to IL-8 by using PMN from a patient with chronic granulomatous disease. PMN constitutively produce adenosine, so we added adenosine deaminase to the transwell assays and observed increased migration to ENA-78 across T84 monolayers. The level of migration was further enhanced by pretreating PMN with adenosine before adding the cells to the assay in the presence of the deaminase. CONCLUSIONS: PMN migration mediated by CXCR2 through the epithelium is regulated by adenosine. Adenosine appears to reduce transepithelial migration by influencing ß2 integrin use on the PMN.

Regulator of calcineurin 1 (Rcan1) is required for the development of pulmonary eosinophilia in allergic inflammation in mice.

The presence of eosinophils in the lung is often regarded as a defining feature of asthma. On allergen stimulation, numbers of eosinophils and their progenitors are increased in both the bone marrow and lungs. Eosinophil progenitors provide an ongoing supply of mature eosinophils. Here, we report that deficiency in the regulator of calcineurin 1 gene (Rcan1) leads to a near-complete absence of eosinophilia in ovalbumin-induced allergic asthma in mice. In the absence of Rcan1, bone marrow cells produce significantly fewer eosinophils in vivo and in vitro on interleukin-5 stimulation. Importantly, eosinophil progenitor populations are significantly reduced in both naïve and ovalbumin-challenged Rcan1(-/-) mice. Bone marrow cells from Rcan1(-/-) mice are capable of developing into fully mature eosinophils, suggesting that Rcan1 is required for eosinophil progenitor production but may not be necessary for eosinophil maturation. Thus, Rcan1 represents a novel contributor in the development of eosinophilia in allergic asthma through regulation of eosinophil progenitor production.

IFN regulatory factor 3 contributes to the host response during Pseudomonas aeruginosa lung infection in mice.

Pseudomonas aeruginosa is a major opportunistic pathogen. However, host defense mechanisms involved in P. aeruginosa lung infection remain incompletely defined. The transcription factor IFN regulatory factor 3 (IRF3) is primarily associated with host defense against viral infections, and a role of IRF3 in P. aeruginosa infection has not been reported previously. In this study, we showed that IRF3 deficiency led to impaired clearance of P. aeruginosa from the lungs of infected mice. P. aeruginosa infection induced IRF3 translocation to the nucleus, activation of IFN-stimulated response elements (ISRE), and production of IFN-beta, suggesting that P. aeruginosa activates the IRF3-ISRE-IFN pathway. In vitro, macrophages from IRF3-deficient mice showed complete inhibition of CCL5 (RANTES) and CXCL10 (IP-10) production, partial inhibition of TNF, but no effect on CXCL2 (MIP-2) or CXCL1 (keratinocyte-derived chemokine) in response to P. aeruginosa stimulation. In vivo, IRF3-deficient mice showed complete inhibition of CCL5 production and partial or no effects on production of other cytokines and chemokines in the bronchoalveolar lavage fluids and lung tissues. Profiling of immune cells in the airways revealed that neutrophil and macrophage recruitment into the airspace was reduced, whereas B cell, T cell, NK cell, and NKT cell infiltration was unaffected in IRF3-deficient mice following P. aeruginosa lung infection. These data suggest that IRF3 regulates a distinct profile of cytokines and chemokines and selectively modulates neutrophil and macrophage recruitment during P. aeruginosa infection. Thus, IRF3 is an integral component in the host defense against P. aeruginosa lung infection.

Depletion of natural CD4+CD25+ T regulatory cells with anti-CD25 antibody does not change the course of Pseudomonas aeruginosa-induced acute lung infection in mice.

Pseudomonas aeruginosa is a common cause of lung infection in immune compromised individuals. Studies in humans and mice have demonstrated that P. aeruginosa lung infection is associated with a predominant Th2 immune response, whereas Th1 responses are accompanied by a better pulmonary outcome. Regulatory T cells (Tregs) are a subpopulation of T cells with unique immunologic characteristics that suppress effector T cell functions. Whether Tregs contribute to P. aeruginosa-induced host responses has not been studied previously. We found that P. aeruginosa lung infection induced an increase in natural Treg cells (CD4+CD25+FOXP3+ T cells) in the spleen of mice. To investigate a role of natural CD4+CD25+ Tregs in the host response to P. aeruginosa lung infection in vivo, anti-CD25 Ab was used to deplete endogenous CD4+CD25+ Tregs. Anti-CD25 treatment depleted 90% of CD4+CD25+FOXP3+ cells. Surprisingly, no differences of P. aeruginosa-induced NF-kappaB activation and cytokine/chemokine production (IL-1beta, TNF, IL-6, IL-10, RANTES or MIP-2) were observed between anti-CD25-treated and isotype control Ab-treated animals. Similarly, no differences in lung histology and airway neutrophil infiltration were observed between anti-CD25 and control Ab-treated animals. Furthermore, no difference in survival outcome was found between anti-CD25 and control Ab-treated animals. These data demonstrate that although P. aeruginosa lung infection causes an increase of Tregs, the endogenous natural CD4+CD25+ Treg cells do not contribute significantly to the host response to this bacterium.

TRAF6 specifically contributes to FcepsilonRI-mediated cytokine production but not mast cell degranulation.

TRAF6 (tumor necrosis factor-associated factor 6) is an essential adaptor downstream from the tumor necrosis factor (TNF) receptor and Toll-like receptor superfamily members. This molecule is critical for dendritic cell maturation and T cell homeostasis. Here we show that TRAF6 is important in high affinity IgE receptor, FcepsilonRI-mediated mast cell activation. In contrast to dendritic cells and T cells, TRAF6-deficient mast cells matured normally and showed normal IgE-dependent degranulation. Importantly, TRAF6-deficient mast cells showed impaired production of cytokine interleukin-6, CCL-9, interleukin-13, and TNF following FcepsilonRI aggregation. Chromatin immunoprecipitation assay showed decreased NF-kappaB p65 binding to CCL-9 and TNF promoters in TRAF6-deficient mast cells. Antigen and IgE-induced IkappaB phosphorylation and NF-kappaB p65 translocation to the nucleus were diminished in TRAF6-deficient mast cells. NF-kappaB luciferase activity in response to antigen and IgE stimulation was severely impaired in TRAF6-deficient mast cells. In addition, antigen and IgE-induced phosphorylation of mitogen-activated protein kinase p38 and JNK, but not ERK1/2, was significantly reduced in TRAF6-deficient mast cells. These results identified TRAF6 as an important signal transducer in FcepsilonRI-mediated signaling in mast cells. Our findings implicate TRAF6 as a new adaptor/regulator molecule for allergen-mediated inflammation in allergy.

Signal transducer and activator of transcription 4 (STAT4), but not IL-12 contributes to Pseudomonas aeruginosa-induced lung inflammation in mice.

Pseudomonas aeruginosa is a major opportunistic pathogen in immune-compromised individuals and cystic fibrosis patients. This organism stimulates a complex inflammatory response in the lung, including production of various cytokines and chemokines. The specific contribution of these mediators in the host defense against this bacterium has yet to be fully characterized. Interleukin-12 (IL-12) is commonly known as a master regulator of innate and adaptive immunity. IL-12 induces its biological effects through its associated intracellular signaling molecule, the signal transducer and activator of transcription 4 (STAT4). To examine a specific role of IL-12 and STAT4 in P. aeruginosa lung infection in mice, STAT4-deficient (STAT4-/-) and IL-12 p40-deficient (IL-12 p40-/-) mice were infected with P. aeruginosa intranasally. Interestingly, STAT4-/- mice, but not IL-12 p40-/- mice after 24h infection showed impaired production of the pro-inflammatory cytokines tumor necrosis factor, interleukin-1beta, and macrophage-inflammatory protein-2. However, neither STAT4 nor IL-12 p40 deficiency significantly affected INFgamma production or bacterial clearance compared to wild-type mice. Similarly, neutrophil recruitment was not affected in the STAT4-/- and IL-12 p40-/- mice. These results suggest that STAT4 contributes to P. aeruginosa-induced inflammation, but it is not essential for bacterial clearance. Although IL-12 is essential for the host defense against various pathogens, this cytokine is likely not a major player in the host response to P. aeruginosa lung infection.

Neutrophil transepithelial migration in response to the chemoattractant fMLP but not C5a is phospholipase D-dependent and related to the use of CD11b/CD18.

In Crohn's disease and ulcerative colitis patients, the numbers of neutrophils recovered from stool directly correlates with the severity of disease, implying that neutrophils in the lumen contribute to the tissue destruction; therefore, it is important to understand the mechanisms behind transintestinal epithelial migration. Neutrophil transintestinal epithelial migration to fMLP is appreciated to be CD11b/CD18 integrin (Mac-1)-dependent, while we recently reported that migration to C5a is Mac-1-independent. Here, we investigated whether phospholipase D (PLD), a signaling molecule linked to chemoattractant activation of neutrophils, is necessary for both Mac-1-dependent and Mac-1-independent migration. Both fMLP and C5a increased neutrophil expression of the Mac-1 activation epitope, indicating PLD was activated. This up-regulation was dose-dependently prevented by incubation of neutrophils in 1-butanol, an inhibitor of PLD activity. Despite this effect on Mac-1, 1-butanol did not prevent neutrophil migration across acellular filters. Incubation in 1-butanol did inhibit fMLP but not C5a-mediated migration across intestinal epithelial cell monolayers, showing that transepithelial migration to fMLP but not C5a is dependent on PLD. The addition of phosphatidic acid, a reaction product of PLD, partially restored fMLP-mediated transepithelial migration in the presence of 1-butanol but not the migration of Mac-1-deficient neutrophil-differentiated HL-60 cells. Thus PLD control over expression of the Mac-1 activation epitope is critical for neutrophil migration to fMLP but not C5a. Moreover, as PLD controls other neutrophil functions, such as the oxidative response, degranulation, and protease release, we could exclude these functions as being important in neutrophil transepithelial migration to C5a.

Neutrophil differentiated HL-60 cells model Mac-1 (CD11b/CD18)-independent neutrophil transepithelial migration.

During active intestinal inflammation granulocytes accumulate in the lumen of the gut where they damage the epithelium through the release of various products such as reactive oxygen species and proteolytic enzymes. Previously, using function blocking monoclonal antibodies, we showed that neutrophil migration across intestinal epithelial monolayers in response to various chemoattractants was partially beta(2) integrin Mac-1 (CD11b/CD18)-independent. Here, we show that treating neutrophils with intact monoclonal antibody (mAb) to CD18 activates the cells to express more CD11b. Thus our goal now was to determine whether neutrophil Mac-1-independent transepithelial migration proceeds independently of prior cell activation through Mac-1. We took two approaches, one using blocking Fab' fragments of mAb to CD18 and the second was to develop a neutrophil differentiated HL-60 cell line which is Mac-1 deficient to further study neutrophil/epithelial cell interaction. Anti-CD18 Fab' minimally activated neutrophils but inhibited approximately 75% of transepithelial migration to fMLP while having a minimal effect (