Thymic stromal lymphopoietin protects in a model of airway damage and inflammation via regulation of caspase-1 activity and apoptosis inhibition.

Thymic stromal lymphopoietin (TSLP), an epithelial cell-derived cytokine, exhibits both pro-inflammatory and pro-homeostatic properties depending on the context and tissues in which it is expressed. It remains unknown whether TSLP has a similar dual role in the airways, where TSLP is known to promote allergic inflammation. Here we show that TSLP receptor (TSLPR)-deficient mice (Tslpr-/-) and mice treated with anti-TSLP antibodies exhibited increased airway inflammation and morbidity rates after bleomycin-induced tissue damage. We found that signaling through TSLPR on non-hematopoietic cells was sufficient for TSLP's protective function. Consistent with this finding, we showed that TSLP reduces caspase-1 and caspase-3 activity levels in primary human bronchial epithelial cells treated with bleomycin via Bcl-xL up-regulation. These observations were recapitulated in vivo by observing that Tslpr-/- mice showed reduced Bcl-xL expression that paralleled increased lung caspase-1 and caspase-3 activity levels and IL-1ß concentrations in the bronchial-alveolar lavage fluid. Our studies reveal a novel contribution for TSLP in preventing damage-induced airway inflammation.

Herpes Virus Entry Mediator (HVEM): A Novel Potential Mediator of Trauma-Induced Immunosuppression.

BACKGROUND: Herpes virus entry mediator (HVEM) is a coinhibitory molecule which can both stimulate and inhibit host immune responses. Altered expression of HVEM and its ligands is associated with increased nosocomial infections in septic patients. We hypothesize critically ill trauma patients will display increased lymphocyte HVEM expression and that such alteration is predictive of infectious events. MATERIALS AND METHODS: Trauma patients prospectively enrolled from the ICU were compared with healthy controls. Leukocytes were isolated from whole blood, stained for CD3 (lymphocytes) and HVEM, and evaluated by flow cytometry. Charts were reviewed for injuries sustained, APACHE II score, hospital course, and secondary infections. RESULTS: Trauma patients (n = 31) were older (46.7 ± 2.4 versus 36.8 ± 2.1 y; P = 0.03) than healthy controls (n = 10), but matched for male sex (74% versus 60%; P = 0.4). Trauma patients had higher presenting WBC (13.9 ± 1.3 versus 5.6 ± 0.5 × 106/mL; P = 0.002), lower percentage of CD3+ lymphocytes (7.5% ± 0.8 versus 22.5% ± 0.9; P < 0.001), but significantly greater expression of HVEM+/CD3+ lymphocytes (89.6% ± 1.46 versus 67.3% ± 1.7; P < 0.001). Among trauma patients, secondary infection during the hospitalization was associated with higher APACHE II scores (20.6 ± 1.6 versus 13.6 ± 1.4; P = 0.03) and markedly lower CD3+ lymphocyte HVEM expression (75% ± 2.6 versus 93% ± 0.7; P < 0.01). CONCLUSIONS: HVEM expression on CD3+ cells increases after trauma. Patients developing secondary infections have less circulating HVEM+CD3+. This implies HVEM signaling in lymphocytes plays a role in maintaining host defense to infection in after trauma. HVEM expression may represent a marker of infectious risk as well as a potential therapeutic target, modulating immune responses to trauma.

Serum Protein Changes in Pediatric Sepsis Patients Identified With an Aptamer-Based Multiplexed Proteomic Approach.

OBJECTIVES: Sepsis, a life-threatening organ dysfunction caused by a dysregulated host response to infection, is a leading cause of death and disability among children worldwide. Identifying sepsis in pediatric patients is difficult and can lead to treatment delay. Our objective was to assess the host proteomic response to infection utilizing an aptamer-based multiplexed proteomics approach to identify novel serum protein changes that might help distinguish between pediatric sepsis and infection-negative systemic inflammation and hence can potentially improve sensitivity and specificity of the diagnosis of sepsis over current clinical criteria approaches. DESIGN: Retrospective, observational cohort study. SETTING: PICU and cardiac ICU, Seattle Children's Hospital, Seattle, WA. PATIENTS: A cohort of 40 children with clinically overt sepsis and 30 children immediately postcardiopulmonary bypass surgery (infection-negative systemic inflammation control subjects) was recruited. Children with sepsis had a confirmed or suspected infection, two or more systemic inflammatory response syndrome criteria, and at least cardiovascular and/or pulmonary organ dysfunction. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Serum samples from 35 of the sepsis and 28 of the bypass surgery subjects were available for screening with an aptamer-based proteomic platform that measures 1,305 proteins to search for large-scale serum protein expression pattern changes in sepsis. A total of 111 proteins were significantly differentially expressed between the sepsis and control groups, using the linear models for microarray data (linear modeling) and Boruta (decision trees) R packages, with 55 being previously identified in sepsis patients. Weighted gene correlation network analysis helped identify 76 proteins that correlated highly with clinical sepsis traits, 27 of which had not been previously reported in sepsis. CONCLUSIONS: The serum protein changes identified with the aptamer-based multiplexed proteomics approach used in this study can be useful to distinguish between sepsis and noninfectious systemic inflammation.

Basophil-derived tumor necrosis factor can enhance survival in a sepsis model in mice.

Basophils are evolutionarily conserved in vertebrates, despite their small numbers and short life span, suggesting that they have beneficial roles in maintaining health. However, these roles are not fully defined. Here we demonstrate that basophil-deficient mice exhibit reduced bacterial clearance and increased morbidity and mortality in the cecal ligation and puncture (CLP) model of sepsis. Among the several proinflammatory mediators that we measured, tumor necrosis factor (TNF) was the only cytokine that was significantly reduced in basophil-deficient mice after CLP. In accordance with that observation, we found that mice with genetic ablation of Tnf in basophils exhibited reduced systemic concentrations of TNF during endotoxemia. Moreover, after CLP, mice whose basophils could not produce TNF, exhibited reduced neutrophil and macrophage TNF production and effector functions, reduced bacterial clearance, and increased mortality. Taken together, our results show that basophils can enhance the innate immune response to bacterial infection and help prevent sepsis.

Respiratory Syncytial Virus Infection of Human Lung Fibroblasts Induces a Hyaluronan-Enriched Extracellular Matrix That Binds Mast Cells and Enhances Expression of Mast Cell Proteases.

Human lung fibroblasts (HLFs) treated with the viral mimetic polyinosine-polycytidylic acid (poly I:C) form an extracellular matrix (ECM) enriched in hyaluronan (HA) that avidly binds monocytes and lymphocytes. Mast cells are important innate immune cells in both asthma and acute respiratory infections including respiratory syncytial virus (RSV); however, the effect of RSV on HA dependent mast cell adhesion and/or function is unknown. To determine if RSV infection of HLFs leads to the formation of a HA-enriched ECM that binds and enhances mast cell activity primary HLFs were infected with RSV for 48 h prior to leukocyte binding studies using a fluorescently labeled human mast cell line (LUVA). Parallel HLFs were harvested for characterization of HA production by ELISA and size exclusion chromatography. In separate experiments, HLFs were infected as above for 48 h prior to adding LUVA cells to HLF wells. Co-cultures were incubated for 48 h at which point media and cell pellets were collected for analysis. The role of the hyaladherin tumor necrosis factor-stimulated gene 6 (TSG-6) was also assessed using siRNA knockdown. RSV infection of primary HLFs for 48 h enhanced HA-dependent LUVA binding assessed by quantitative fluorescent microscopy. This coincided with increased HLF HA synthase (HAS) 2 and HAS3 expression and decreased hyaluronidase (HYAL) 2 expression leading to increased HA accumulation in the HLF cell layer and the presence of larger HA fragments. Separately, LUVAs co-cultured with RSV-infected HLFs for 48 h displayed enhanced production of the mast cell proteases, chymase, and tryptase. Pre-treatment with the HA inhibitor 4-methylumbelliferone (4-MU) and neutralizing antibodies to CD44 (HA receptor) decreased mast cell protease expression in co-cultured LUVAs implicating a direct role for HA. TSG-6 expression was increased over the 48-h infection. Inhibition of HLF TSG-6 expression by siRNA knockdown led to decreased LUVA binding suggesting an important role for this hyaladherin for LUVA adhesion in the setting of RSV infection. In summary, RSV infection of HLFs contributes to inflammation via HA-dependent mechanisms that enhance mast cell binding as well as mast cell protease expression via direct interactions with the ECM.

A Potential Mechanism for Immune Suppression by Beta-Adrenergic Receptor Stimulation following Traumatic Injury.

BACKGROUND: ß-Adrenergic agents suppress inflammation and may play an important role in posttraumatic infections. Mechanisms may include inhibition of MAP kinase signaling. We sought to determine whether MKP-1 contributed to catecholamine suppression of innate immunity and also wanted to know whether early catecholamine treatment after traumatic injury increases the risk of later nosocomial infection. METHODS: We performed experiments using THP-1 cells and peripheral blood mononuclear cells from healthy individuals. We exposed cells to epinephrine and/or LPS and measured inflammatory gene transcription and MAP kinase activation. We inhibited MKP-1 activity to determine its role in catecholamine-induced immune suppression. Finally, we studied injured subjects to determine whether early catecholamine treatment was associated with nosocomial infection. RESULTS: Epinephrine increases MKP-1 transcripts and protein and decreases LPS-induced p38 and JNK phosphorylation and TNF-α gene transcription. RNAi inhibition of MKP-1 at least partially restores LPS-induced TNF-α gene expression (p = 0.024). In the clinical cohort, subjects treated with ß-adrenergic agents had an increased risk of ventilator-associated pneumonia (aOR = 1.9; 95% CI = 1.3-2.6) and bacteremia (aOR = 1.5; 95% CI = 1.1-2.3). CONCLUSIONS: MKP-1 may have a role in catecholamine-induced suppression of innate immunity, and exogenous catecholamines might contribute to nosocomial infection risk.

Proteome analysis of mast cell releasates reveals a role for chymase in the regulation of coagulation factor XIIIA levels via proteolytic degradation.

BACKGROUND: Mast cells are significantly involved in IgE-mediated allergic reactions; however, their roles in health and disease are incompletely understood. OBJECTIVE: We aimed to define the proteome contained in mast cell releasates on activation to better understand the factors secreted by mast cells that are relevant to the contribution of mast cells in diseases. METHODS: Bone marrow-derived cultured mast cells (BMCMCs) and peritoneal cell-derived mast cells were used as "surrogates" for mucosal and connective tissue mast cells, respectively, and their releasate proteomes were analyzed by mass spectrometry. RESULTS: Our studies showed that BMCMCs and peritoneal cell-derived mast cells produced substantially different releasates following IgE-mediated activation. Moreover, we observed that the transglutaminase coagulation factor XIIIA (FXIIIA) was one of the most abundant proteins contained in the BMCMC releasates. Mast cell-deficient mice exhibited increased FXIIIA plasma and activity levels as well as reduced bleeding times, indicating that mast cells are more efficient in their ability to downregulate FXIIIA than in contributing to its amounts and functions in homeostatic conditions. We found that human chymase and mouse mast cell protease-4 (the mouse homologue of human chymase) had the ability to reduce FXIIIA levels and function via proteolytic degradation. Moreover, we found that chymase deficiency led to increased FXIIIA amounts and activity, as well as reduced bleeding times in homeostatic conditions and during sepsis. CONCLUSIONS: Our study indicates that the mast cell protease content can shape its releasate proteome. Moreover, we found that chymase plays an important role in the regulation of FXIIIA via proteolytic degradation.

Thymic Stromal Lymphopoietin Improves Survival and Reduces Inflammation in Sepsis.

The mechanisms that contribute to homeostasis of the immune system in sepsis are largely unknown. One study suggests a potential detrimental role for thymic stromal lymphopoietin (TSLP) in sepsis; however, the immune-regulatory effects of TSLP on myeloid cells within the intestinal microenvironment suggest the contrary. Our objective was to clarify TSLP's role in sepsis. Cecal ligation and puncture was performed in mice with total or myeloid-specific deficiency in the TSLP receptor (TSLPR). Survival was monitored closely, peritoneal fluids and plasma were analyzed for markers of inflammation, and myeloid cell numbers and their ability to produce inflammatory mediators was determined. The interaction of TSLP with TSLPR in myeloid cells contributed to mouse survival after septic peritonitis. Mice with TSLPR deficiency in myeloid cells displayed excessive local and systemic inflammation levels (e.g., increased inflammatory cell and cytokine levels) relative to control mice. Moreover, hepatic injury was exacerbated in mice with TSLPR deficiency in their myeloid cells. However, the enhanced inflammatory response did not affect the ability of these mice to clear bacteria. Resident neutrophils and macrophages from septic mice with TSLPR deficiency exhibited an increased ability to produce proinflammatory cytokines. Collectively, our findings suggest that the effects of TSLP on myeloid cells are crucial in reducing the multiple organ failure that is associated with systemic inflammation, which highlights the significance of this cytokine in modulating the host response to infection and in reducing the risks of sepsis development.

BTLA expression contributes to septic morbidity and mortality by inducing innate inflammatory cell dysfunction.

A proper innate inflammatory response is essential for prevention of the systemic inflammation associated with sepsis. BTLA is an immune-regulatory receptor demonstrated to be expressed not only on adaptive immune populations and have potent inhibitory effects on CD4(+) T cells but is also expressed on innate cell populations (CD11c(+) and CD11b(+) cells) and has been shown to diminish pathogen clearance following bacterial and parasite infection. The role of BTLA in sepsis and the mechanisms by which BTLA alters pathogen clearance, however, have not been addressed clearly. Here, we show that following acute experimental sepsis induction in mice (CLP), the number of infiltrating BTLA- and HVEM (the ligand for BTLA)-expressing macrophages, inflammatory monocytes, mature and immature DCs, and neutrophils increased in the peritoneum compared with sham surgery, suggesting that a high level of HVEM:BTLA interactions occurs between these cells at the site of septic insult. Given this, we evaluated BTLA(-/-) mice, 24 h post-CLP, and observed a marked increase in the degree of activation on these cell populations, as well as a reduction in peritoneal bacterial burden and IL-10 induction, and most importantly, BTLA(-/-) mice exhibited a higher rate of survival and protection from organ injury when compared with WT mice. Such changes were not restricted to experimental mice, as circulating BTLA+ and HVEM+ monocytes and HVEM+ granulocytes were increased in septic ICU patients, supporting a role for BTLA and/or HVEM as potential, novel diagnostic markers of innate immune response/status and as therapeutic targets of sepsis.