Pulmonary Hemorrhage in Pediatric Hematopoietic Stem Cell Transplant Recipients: A Single-Center Retrospective Study.

OBJECTIVES: Pulmonary hemorrhage (PH) is a serious complication posthematopoietic stem cell transplant (HSCT). In view of limited available pediatric data, we performed a retrospective study to describe epidemiology, management, and outcomes of PH post-HSCT in children in our national center. DESIGN: Retrospective study. SETTING: Academic children's hospital (2000-2015). SUBJECTS: Children (< 18 yr) with PH and requiring PICU care post-HSCT. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The historical prevalence of PH in our center was 2.7% (31/1,148). Twenty patients had a concomitant infection, 15 had bacterial infection, 8 had viral infection, and 3 patients had a fungal infection. With a median follow-up time of 60 months, 7 of 31 patients were alive. Early PH (< 40 d post-HSCT) was associated with improved survival (6/15 vs 1/16, p = 0.035). Patients who received high-dose pulsed corticosteroid had improved survival when compared with those who did not (7/22 vs 0/9, p = 0.0012); this also applied to the subgroup of patients with a concomitant infection (5/15 vs 0, p = 0.001). None of the patients who survived had measurable respiratory sequelae. CONCLUSIONS: PH is a rare but serious complication after HSCT. Corticosteroids were associated with improved survival even in patients with a concomitant infection.

Comparative analysis of respiratory symptom scores to detect acute respiratory events in children with cystic fibrosis.

BACKGROUND: Patient-reported outcomes (PROs) are important outcome measures in research and clinical practice. This study describes the longitudinal variability the Cystic Fibrosis Questionnaire-Revised (CFQ-R) Respiratory score and the Chronic Respiratory Infection Symptom Score (CRISS), as well as their ability to identify acute respiratory events in children with CF. METHODS: In this prospective observational study, the parent-proxy (6 -13 years) and self-reported (6-18 years) CFQ-R Respiratory score and CRISS (6-18 years) were measured every 3 months over 2 years. The lung clearance index (LCI) and FEV1 were also measured. We compared the diagnostic accuracy of the PROs in distinguishing acute respiratory events and clinically stable visits, using the minimal important difference of each PRO as the threshold. RESULTS: A total of 98 children with CF were included. On average, the symptom scores did not change between clinically stable visits. The positive predictive value (PPV) and negative predictive value (NPV) of a ≥8.5-point worsening in the parent-proxy CFQ-R score to identify acute respiratory events (n=119) (PPV 70.2% and NPV 87.0%) were higher than for the self-reported CFQ-R score (PPV 58.9% and NPV 72.2%). The PPV and NPV of an ≥11-point change in the CRISS for acute respiratory events (n=137) was 56.5% and 79.6%, respectively. The PPV and NPV of all PROs were increased when combined with the LCI and/or FEV1pp. CONCLUSION: Symptoms scores differ in their ability to identify acute respiratory events in children with CF; PPV and NPV of all PROs were improved when combined with lung function outcomes.

Evaluation of clinically relevant changes in the lung clearance index in children with cystic fibrosis and healthy controls.

BACKGROUND: The limits of reproducibility of the lung clearance index (LCI) are higher in children with cystic fibrosis (CF) compared with healthy children, and it is currently unclear what defines a clinically meaningful change. METHODS: In a prospective multisite observational study of children with CF and healthy controls (HCs), we measured LCI, FEV1% predicted and symptom scores at quarterly visits over 2 years. Two reviewers performed a detailed review of visits to evaluate the frequency that between visit LCI changes outside ±10%, ±15%, ±20% represented a clinically relevant signal. In the setting of acute respiratory symptoms, we used a generalised estimating equation model, with a logit link function to determine the ability of LCI worsening at different thresholds to predict failure of lung function recovery at follow-up. RESULTS: Clinically relevant LCI changes outside ±10%, ±15% and ±20% were observed at 25.7%, 15.0% and 8.3% of CF visits (n=744), respectively. The proportions of LCI changes categorised as noise, reflecting biological variability, were comparable between CF and HC at the 10% (CF 9.9% vs HC 13.0%), 15% (CF 4.3% vs HC 3.1%) and 20% (CF 2.4% vs HC 1.0%) thresholds. Compared with symptomatic CF visits without a worsening in LCI, events with ≥10% LCI increase were more likely to fail to recover baseline LCI at follow-up. CONCLUSION: The limits of reproducibility of the LCI in healthy children can be used to detect clinically relevant changes and thus inform clinical care in children with CF.

Determinants of lung disease progression measured by lung clearance index in children with cystic fibrosis.

The lung clearance index (LCI) measured by the multiple breath washout (MBW) test is sensitive to early lung disease in children with cystic fibrosis. While LCI worsens during the preschool years in cystic fibrosis, there is limited evidence to clarify whether this continues during the early school age years, and whether the trajectory of disease progression as measured by LCI is modifiable.A cohort of children (healthy and cystic fibrosis) previously studied for 12 months as preschoolers were followed during school age (5-10 years). LCI was measured every 3 months for a period of 24 months using the Exhalyzer D MBW nitrogen washout device. Linear mixed effects regression was used to model changes in LCI over time.A total of 582 MBW measurements in 48 healthy subjects and 845 measurements in 64 cystic fibrosis subjects were available. The majority of children with cystic fibrosis had elevated LCI at the first preschool and first school age visits (57.8% (37 out of 64)), whereas all but six had normal forced expiratory volume in 1 s (FEV1) values at the first school age visit. During school age years, the course of disease was stable (-0.02 units·year-1 (95% CI -0.14-0.10). LCI measured during preschool years, as well as the rate of LCI change during this time period, were important determinants of LCI and FEV1, at school age.Preschool LCI was a major determinant of school age LCI; these findings further support that the preschool years are critical for early intervention strategies.

Lung Clearance Index to Track Acute Respiratory Events in School-Age Children with Cystic Fibrosis.

Rationale: The lung clearance index (LCI) is responsive to acute respiratory events in preschool children with cystic fibrosis (CF), but its utility to identify and manage these events in school-age children with CF is not well defined.Objectives: To describe changes in LCI with acute respiratory events in school-age children with CF.Methods: In a multisite prospective observational study, the LCI and FEV1 were measured quarterly and during acute respiratory events. Linear regression was used to compare relative changes in LCI and FEV1% predicted at acute respiratory events. Logistic regression was used to compare the odds of a significant worsening in LCI and FEV1% predicted at acute respiratory events. Generalized estimating equation models were used to account for repeated events in the same subject.Measurements and Main Results: A total of 98 children with CF were followed for 2 years. There were 265 acute respiratory events. Relative to a stable baseline measure, LCI (+8.9%; 95% confidence interval, 6.5 to 11.3) and FEV1% predicted (-6.6%; 95% confidence interval, -8.3 to -5.0) worsened with acute respiratory events. A greater proportion of events had a worsening in LCI compared with a decline in FEV1% predicted (41.7% vs. 30.0%; P = 0.012); 53.9% of events were associated with worsening in LCI or FEV1. Neither LCI nor FEV1 recovered to baseline values at the next follow-up visit.Conclusions: In school-age children with CF, the LCI is a sensitive measure to assess lung function worsening with acute respiratory events and incomplete recovery at follow-up. In combination, the LCI and FEV1 capture a higher proportion of events with functional impairment.

Progression of Cystic Fibrosis Lung Disease from Childhood to Adulthood: Neutrophils, Neutrophil Extracellular Trap (NET) Formation, and NET Degradation.

Genetic defects in cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene cause CF. Infants with CFTR mutations show a peribronchial neutrophil infiltration prior to the establishment of infection in their lung. The inflammatory response progressively increases in children that include both upper and lower airways. Infectious and inflammatory response leads to an increase in mucus viscosity and mucus plugging of small and medium-size bronchioles. Eventually, neutrophils chronically infiltrate the airways with biofilm or chronic bacterial infection. Perpetual infection and airway inflammation destroy the lungs, which leads to increased morbidity and eventual mortality in most of the patients with CF. Studies have now established that neutrophil cytotoxins, extracellular DNA, and neutrophil extracellular traps (NETs) are associated with increased mucus clogging and lung injury in CF. In addition to opportunistic pathogens, various aspects of the CF airway milieux (e.g., airway pH, salt concentration, and neutrophil phenotypes) influence the NETotic capacity of neutrophils. CF airway milieu may promote the survival of neutrophils and eventual pro-inflammatory aberrant NETosis, rather than the anti-inflammatory apoptotic death in these cells. Degrading NETs helps to manage CF airway disease; since DNAse treatment release cytotoxins from the NETs, further improvements are needed to degrade NETs with maximal positive effects. Neutrophil-T cell interactions may be important in regulating viral infection-mediated pulmonary exacerbations in patients with bacterial infections. Therefore, clarifying the role of neutrophils and NETs in CF lung disease and identifying therapies that preserve the positive effects of neutrophils, while reducing the detrimental effects of NETs and cytotoxic components, are essential in achieving innovative therapeutic advances.

A dual neutrophil-T cell purification procedure and methodological considerations in studying the effects of estrogen on human Th17 cell differentiation.

New procedures are required to optimize the use of blood samples to study different cell types. The purification of neutrophils and T cells from the same blood sample is not commonly described. We have previously used PolymorphPrep™ (P) or LymphoPrep™ (L) for purifying neutrophils or T cells, respectively. In this study, we describe a new method for purifying both of these cells using P and L from the same sample, and methodological considerations required to obtain consistent Th17 differentiation results. For T cell studies, we first isolated mononuclear cells from peripheral blood of healthy humans using either P alone, L alone or sequential isolation with P and then L (P + L). CD3+ lymphocytes comprise up to 73% of peripheral blood mononuclear cells (PBMCs) obtained by sequential isolation, with 29% and 36% for P and L, respectively. T lymphocyte subsets, Th1, Th17 or double-positive (Th17/1), were then amplified. Four days of amplification culture after isolation by P alone led to over-expression of Th17/1 cells and of Th17 cells in comparison to cells isolated by L or by sequential P + L. Th17/1 cells comprised 11.0 ±â€¯6.8% (P alone) vs 1.2 ±â€¯0.28% (L alone) vs 0.45 ±â€¯0.11% (P + L) and Th17 cells comprised 2.8 ±â€¯0.4% (P alone) 0.88 ±â€¯0.15% (L alone) vs 0.86 ±â€¯0.14% (P + L). As the second step, we examined T cell purification and differentiation. A higher purity of 97.1 ±â€¯0.44% naïve CD4+ T cell was reached after P + L followed by immunomagnetic bead sorting in comparison to 70 ±â€¯9.3% (L) vs 21.0 ±â€¯8.5% (P). These cells grew well in the density range of 25, 000 to 100, 000 cells per well in 96-well plates during Th17 cell differentiation; higher or lower cell density did not support Th17 cell differentiation. Lastly, to investigate the effect of estrogen on Th17 cell differentiation, serum-free AIM V medium without phenol red was chosen to minimize the hormonal effects of the medium. We found that exogenous estrogen (1 nM) inhibited Th17 cell differentiation in this medium. Taken together, we devised a method to isolate both neutrophils and T cells from the same blood sample and show that high PBMC purity, selected culture medium and an optimal cell density of the initial cell culture produced the most robust and consistent results for Th17 differentiation.

Two-in-one: UV radiation simultaneously induces apoptosis and NETosis.

NETosis is a unique form of neutrophil death that differs from apoptosis and necrosis. However, whether NETosis and apoptosis can occur simultaneously in the same neutrophil is unknown. In this paper, we show that increasing doses of ultraviolet (UV) irradiation increases NETosis, which is confirmed by myeloperoxidase colocalisation to neutrophil extracellular DNA. Increasing UV irradiation increases caspase 3 activation, mitochondrial reactive oxygen species (ROS) generation and p38, but not ERK, phosphorylation. Inhibition of mitochondrial ROS production and p38 activation, but not NADPH oxidase (NOX) activity, suppresses UV-induced NETosis, indicating that UV induces NOX-independent NETosis. Like classical NOX-dependent and -independent NETosis, UV-induced NETosis requires transcriptional firing for chromatin decondensation. Cell death-specific inhibitor studies indicate that UV-mediated NETosis is not apoptosis, necrosis or necroptosis. Collectively, these studies indicate that increasing doses of UV irradiation induce both apoptosis and NETosis simultaneously, but the ultimate outcome is the induction of a novel form of NOX-independent NETosis, or "ApoNETosis".

Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis.

Neutrophils migrating from the blood (pH 7.35-7.45) into the surrounding tissues encounter changes in extracellular pH (pHe) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H+ ions reducing the intracellular pH (pHi). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pHe (ranging from 6.6 to 7.8; every 0.2 units) increased pHi of both activated and resting neutrophils within 10-20 min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H+ ions, pHi is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pHe promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pHe-mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H+ ions, whereas each bicarbonate HCO3- ion binds 1H+ ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar bicarbonate solution. For that reason, regulating NETosis by pH with specific buffers such as THAM could be more effective than bicarbonate in managing NET-related diseases.

Efficacy of high-dose steroids for bronchiolitis obliterans syndrome post pediatric hematopoietic stem cell transplantation.

BOS is the pulmonary manifestation of cGvHD post-allogeneic HSCT. Survival and treatment of this often fatal complication have not improved over the last 20 years and there is no clear standard of care. For the past 10 years, BOS was treated in our center with monthly cycles of HDPS. We reviewed the outcomes of patients with post-HSCT BOS who met the diagnostic criteria for BOS as per the NIH consensus and were treated with at least one cycle of methylprednisolone at a dose of 10-30 mg/kg/d×3 d. We collected demographic and clinical data, responses to treatment and results of pulmonary function tests at several time points. Between January 2007 and January 2014, 12 patients were treated with HDPS for post-HSCT BOS. Five patients (42%) had a good response to treatment; four patients (33%) stabilized with moderate lung disease; and three patients (25%) progressed to end-stage disease. No significant acute side effects attributable to the HDPS treatment were identified. HDPS may be an effective treatment option for all but the most severely ill patients with post-HSCT BOS.

Surfactant protein D regulates caspase-8-mediated cascade of the intrinsic pathway of apoptosis while promoting bleb formation.

Surfactant-associated protein D (SP-D) is a soluble innate immune collectin present on many mucosal surfaces. We recently showed that SP-D suppresses the extrinsic pathway of apoptosis by downregulating caspase-8 activation. However, the effects of SP-D on the intrinsic pathway of apoptosis are not clearly understood. In the intrinsic pathway, cytochrome c is released by mitochondria into the cytoplasm. Oxidation of cytochrome c by cytochrome c oxidase activates the apoptosome and caspase-9 cascade. Both caspase-8- and caspase-9-mediated branches are activated in the intrinsic pathway of apoptosis; however, little is known about the relevance of the caspase-8 pathway in this context. Here we studied the effects of SP-D on different branches of the intrinsic pathway of apoptosis using UV-irradiated Jurkat T-cells. We found that SP-D does not inhibit the caspase-9 branch of apoptosis and the relevance of the caspase-8-related branch became apparent when the caspase-9 pathway was inhibited by blocking cytochrome c oxidase. Under these conditions, SP-D reduces the activation of caspase-8, executioner caspase-3 and exposure of phosphatidylserine (PS) on the membranes of dying cells. By contrast, SP-D increases the formation of nuclear and membrane blebs. Inhibition of caspase-8 confirms the effect of SP-D is unique to the caspase-8 pathway. Overall, SP-D suppresses certain aspects of the intrinsic pathway of apoptosis via reduction of caspase-8 activation and PS flipping while at the same time increasing membrane and nuclear bleb formation. This novel regulatory aspect of SP-D could help to regulate intrinsic pathway of apoptosis to promote effective blebbing and breakdown of dying cells.

JNK Activation Turns on LPS- and Gram-Negative Bacteria-Induced NADPH Oxidase-Dependent Suicidal NETosis.

Neutrophils cast neutrophil extracellular traps (NETs) to ensnare microbial pathogens. Nevertheless, the molecular rheostats that regulate NETosis in response to bacteria are not clearly established. We hypothesized that stress-activated protein kinase or c-Jun N-terminal Kinase (SAPK/JNK) is a molecular switch that turns on NETosis in response to increasing concentrations of lipopolysaccharide (LPS)- and Gram-negative bacteria. Here we show that Escherichia coli LPS (0111:B4; 10-25 µg/ml), but not phorbol myristate acetate (PMA), activates JNK in human neutrophils in a dose-dependent manner. JNK inhibitors SP600125 and TCSJNK6o, and a TLR4 inhibitor TAK242 suppress reactive oxygen species production and NETosis in LPS-, but not PMA-treated neutrophils. Diphenyleneiodonium suppresses LPS-induced NETosis, confirming that endotoxin induces NADPH oxidase-dependent NETosis. Immunoblots, Sytox Green assays, and confocal microscopy of cleaved caspase-3 and nuclear morphology show that JNK inhibition does not induce apoptosis in LPS-stimulated neutrophils. JNK inhibition also suppresses NETosis induced by two typical Gram-negative bacteria, E. coli and Pseudomonas aeruginosa. Therefore, we propose that neutrophils use a TLR4-dependent, JNK-mediated molecular sensing mechanism to initiate NADPH oxidase-dependent suicidal NETosis in response to increasing concentrations of LPS, and Gram-negative bacteria. The LPS-TLR4-JNK activation axis determines the fate of these cells: to be or not to be NETotic neutrophils.

Ultraviolet irradiation increases green fluorescence of dihydrorhodamine (DHR) 123: false-positive results for reactive oxygen species generation.

Dihydrorhodamine (DHR) 123 is a fluorophore commonly used for measuring reactive oxygen species (ROS), often after exposing cells to ultraviolet (UV) irradiation or oxidative burst inducers such as Phorbol 12-myristate 13-acetate (PMA). However, the negative effects of UV irradiation on oxidation of DHR123 itself to green fluorescence rhodamine (R) 123 under different experimental conditions (e.g., different buffers, media, cells, ROS detection techniques) have not been fully appreciated. We determined the effect of UV on DHR123 fluorescence, using a cell-free system, and A549 epithelial cells, NIH/3T3 fibroblast cells, Jurkat T cells, primary human T cells, HL-60 neutrophils and primary human neutrophils. We found that UV irradiation rapidly increases green fluorescence of DHR123 in cell-free solutions. The intensity of green fluorescence increases with increasing amounts of DHR123 and UV exposure. The fluorescence increase was greater in Roswell Park Memorial Institute medium (RPMI) than DMEM media. The presence of DMSO (0-1.25%, v/v) in RPMI further increases the fluorescence signal. Phosphate buffered solution (PBS) and Hanks' Balanced Salt Solution (HBSS) generate considerable background signal with DHR123, and increasing DMSO concentration greatly increases the fluorescence signal in these buffers. However, after UV irradiation the amount of DHR123 that remains unoxidized generates sufficient fluorescence signal to measure the ROS produced by H2O2 and peroxidase in vitro or Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated ROS production within HL-60 neutrophils or primary human neutrophils. We conclude that UV irradiation oxidizes DHR123 to generate Rhodamine 123 (R123) green fluorescence signal, and that the R123 present in the culture supernatant could give erroneous results in plate reader assays. However, flow cytometry and fluorescence microscopy reliably detect ROS in cells such as neutrophils. Overall, avoiding false-positive results when detecting ROS using DHR123 requires selection of, agonists, the correct buffers, media, cell types, and measurement techniques.

Surfactant protein D delays Fas- and TRAIL-mediated extrinsic pathway of apoptosis in T cells.

Only a few extracellular soluble proteins are known to modulate apoptosis. We considered that surfactant-associated protein D (SP-D), an innate immune collectin present on many mucosal surfaces, could regulate apoptosis. Although SP-D is known to be important for immune cell homeostasis, whether SP-D affects apoptosis is unknown. In this study we aimed to determine the effects of SP-D on Jurkat T cells and human T cells dying by apoptosis. Here we show that SP-D binds to Jurkat T cells and delays the progression of Fas (CD95)-Fas ligand and TRAIL-TRAIL receptor induced, but not TNF-TNF receptor-mediated apoptosis. SP-D exerts its effects by reducing the activation of initiator caspase-8 and executioner caspase-3. SP-D also delays the surface exposure of phosphatidylserine. The effect of SP-D was ablated by the presence of caspase-8 inhibitor, but not by intrinsic pathway inhibitors. The binding ability of SP-D to dying cells decreases during the early stages of apoptosis, suggesting the release of apoptotic cell surface targets during apoptosis. SP-D also delays FasL-induced death of primary human T cells. SP-D delaying the progression of the extrinsic pathway of apoptosis could have important implications in regulating immune cell homeostasis at mucosal surfaces.

Alkaline pH Promotes NADPH Oxidase-Independent Neutrophil Extracellular Trap Formation: A Matter of Mitochondrial Reactive Oxygen Species Generation and Citrullination and Cleavage of Histone.

pH is highly variable in different tissues and affects many enzymatic reactions in neutrophils. In response to calcium ionophores such as A23187 and ionomycin, neutrophils undergo nicotinamide adenine dinucleotide phosphate oxidase (NOX)-independent neutrophil extracellular trap (NET) formation (NETosis). However, how pH influences calcium-dependent Nox-independent NET formation is not well understood. We hypothesized that increasing pH promotes Nox-independent NET formation by promoting calcium influx, mitochondrial reactive oxygen species (mROS) generation, histone citrullination, and histone cleavage. Here, we show that stimulating human neutrophils isolated from peripheral blood with calcium ionophore A23187 or ionomycin in the media with increasing extracellular pH (6.6, 6.8, 7.0, 7.2, 7.4, 7.8) drastically increases intracellular pH within in 10-20 min. These intracellular pH values are much higher compared to unstimulated cells placed in the media with corresponding pH values. Raising pH slightly drastically increases intracellular calcium concentration in resting and stimulated neutrophils, respectively. Like calcium, mROS generation also increases with increasing pH. An mROS scavenger, MitoTempo, significantly suppresses calcium ionophore-mediated NET formation with a greater effect at higher pH, indicating that mROS production is at least partly responsible for pH-dependent suppression of Nox-independent NETosis. In addition, raising pH increases PAD4 activity as determined by the citrullination of histone (CitH3) and histone cleavage determined by Western blots. The pH-dependent histone cleavage is reproducibly very high during ionomycin-induced NETosis compared to A23187-induced NETosis. Little or no histone cleavage was noted in unstimulated cells, at any pH. Both CitH3 and cleavage of histones facilitate DNA decondensation. Therefore, alkaline pH promotes intracellular calcium influx, mROS generation, PAD4-mediated CitH3 formation, histone 4 cleavage and eventually NET formation. Calcium-mediated NET formation and CitH3 formation are often related to sterile inflammation. Hence, understanding these important mechanistic steps helps to explain how pH regulates NOX-independent NET formation, and modifying pH may help to regulate NET formation during sterile inflammation or potential damage caused by compounds such as ionomycin, secreted by Streptomyces, a group of Gram-positive bacteria well known for producing antibiotics.

CRISPLD2 (LGL1) inhibits proinflammatory mediators in human fetal, adult, and COPD lung fibroblasts and epithelial cells.

Chronic lung disease of prematurity/bronchopulmonary dysplasia (BPD) is the leading cause of perinatal morbidity in developed countries. Inflammation is a prominent finding. Currently available interventions have associated toxicities and limited efficacy. While BPD often resolves in childhood, survivors of preterm birth are at risk for acquired respiratory disease in early life and are more likely to develop chronic obstructive pulmonary disease (COPD) in adulthood. We previously cloned Crispld2 (Lgl1), a glucocorticoid-regulated mesenchymal secretory protein that modulates lung branching and alveogenesis through mesenchymal-epithelial interactions. Absence of Crispld2 is embryonic lethal. Heterozygous Crispld2+/- mice display features of BPD, including distal airspace enlargement, disruption of elastin, and neonatal lung inflammation. CRISPLD2 also plays a role in human fetal lung fibroblast cell expansion, migration, and mesenchymal-epithelial signaling. This study assessed the effects of endogenous and exogenous CRISPLD2 on expression of proinflammatory mediators in human fetal and adult (normal and COPD) lung fibroblasts and epithelial cells. CRISPLD2 expression was upregulated in a lipopolysaccharide (LPS)-induced human fetal lung fibroblast line (MRC5). LPS-induced upregulation of the proinflammatory cytokines IL-8 and CCL2 was exacerbated in MRC5-CRISPLD2(knockdown) cells. siRNA suppression of endogenous CRISPLD2 in adult lung fibroblasts (HLFs) led to augmented expression of IL-8, IL-6, CCL2. LPS-stimulated expression of proinflammatory mediators by human lung epithelial HAEo- cells was attenuated by purified secretory CRISPLD2. RNA sequencing results from HLF-CRISPLD2(knockdown) suggest roles for CRISPLD2 in extracellular matrix and in inflammation. Our data suggest that suppression of CRISPLD2 increases the risk of lung inflammation in early life and adulthood.

LGL1 modulates proliferation, apoptosis, and migration of human fetal lung fibroblasts.

Rapid growth and formation of new gas exchange units (alveogenesis) are hallmarks of the perinatal lung. Bronchopulmonary dysplasia (BPD), common in very premature infants, is characterized by premature arrest of alveogenesis. Mesenchymal cells (fibroblasts) regulate both lung branching and alveogenesis through mesenchymal-epithelial interactions. Temporal or spatial deficiency of late-gestation lung 1/cysteine-rich secretory protein LD2 (LGL1/CRISPLD2), expressed in and secreted by lung fibroblasts, can impair both lung branching and alveogenesis (LGL1 denotes late gestation lung 1 protein; LGL1 denotes the human gene; Lgl1 denotes the mouse/rat gene). Absence of Lgl1 is embryonic lethal. Lgl1 levels are dramatically reduced in oxygen toxicity rat models of BPD, and heterozygous Lgl1(+/-) mice exhibit features resembling human BPD. To explore the role of LGL1 in mesenchymal-epithelial interactions in developing lung, we developed a doxycycline (DOX)-inducible RNA-mediated LGL1 knockdown cellular model in human fetal lung fibroblasts (MRC5(LGL1KD)). We assessed the impact of LGL1 on cell proliferation, cell migration, apoptosis, and wound healing. DOX-induced MRC5(LGL1KD) suppressed cell growth and increased apoptosis of annexin V(+) staining cells and caspase 3/7 activity. LGL1-conditioned medium increased migration of fetal rat primary lung epithelial cells and human airway epithelial cells. Impaired healing by MRC5(LGL1KD) cells of a wound model was attenuated by addition of LGL1-conditioned medium. Suppression of LGL1 was associated with dysregulation of extracellular matrix genes (downregulated MMP1, ColXVα1, and ELASTIN) and proapoptosis genes (upregulated BAD, BAK, CASP2, and TNFRSF1B) and inhibition of 44/42MAPK phosphorylation. Our findings define a role for LGL1 in fibroblast expansion and migration, epithelial cell migration, and mesenchymal-epithelial signaling, key processes in fetal lung development.

T cell unresponsiveness in a pediatric cystic fibrosis patient: a case report.

A girl was diagnosed with cystic fibrosis (CF) at birth, with repeatedly positive sweat tests and homozygous F508del mutations of her CF transmembrane conductance regulator (CFTR) gene. From an early age, her lung disease was more severe than her birth cohort peers despite aggressive treatment. At the age of 16 she was listed for lung transplantation, but prior to transplant was not on systemic corticosteroids or other immunosuppressive agents. In response to ex vivo stimulation, her pre-transplant peripheral blood T cells unexpectedly failed to produce detectable levels of IFN-γ, unlike cells from healthy controls or from another girl with CF and lung disease of comparable severity. Furthermore, naïve T cells freshly isolated from her peripheral blood showed a complete block of T cell differentiation into Th1, Th17 and Treg lineages, even in the presence of cytokines known to promote differentiation into the respective lineages. Her serology has been remarkably devoid of evidence of exposure to viruses that have been associated with T cell exhaustion. However, her freshly isolated naïve T cells showed sustained expression of markers of T cell exhaustion, which were further induced upon ex vivo stimulation, pointing to T cell exhaustion as the cause of the failure of naïve T cells to undergo differentiation in response to cytokine stimulation. Although excessive inflammation in CF lung can be both ineffective at clearing certain pathogens as well as destructive to the lung tissue itself, adequate inflammation is a component of an effective overall immune response to microbial pathogens. Our present findings suggest that intrinsic impairment of T cell differentiation may have contributed to the greater severity and more rapid progression of her CF lung disease than of the lung disease of most of her peers.

The cystic fibrosis gender gap: potential roles of estrogen.

Cystic fibrosis (CF) is a complex, multi-system, autosomal recessive disease predominantly affecting Caucasians that leads to vigorous airway inflammation and chronic respiratory infection, commonly with Pseudomonas aeruginosa. A variety of factors significantly modify the progression and severity of CF lung disease and the timing of the resulting mortality. We summarize here data indicating that there is in CF a female disadvantage in survival and morbidity, called the "CF gender gap". Although controversy exists regarding the nature and relative importance of the various contributing mechanisms involved, gender affects the progression of CF disease with respect to lung infection, decline in pulmonary function and nutritional status. These interrelated factors in turn have a negative impact on survival. This review will emphasize the increasing evidence that suggest a role for the effects of gender, and particularly the female sex hormone estrogen, on infection, inflammation and transepithelial ion transport, all major determinants of CF lung disease. Future elucidation of the pathophysiology of hormonal aggravation of CF lung disease may pave the way for novel therapeutic interventions. This, combined with the magnitude of the gender gap in CF mortality, strongly suggests that further work in this field is well justified.

Intrinsic predisposition of naïve cystic fibrosis T cells to differentiate towards a Th17 phenotype.

BACKGROUND: Cystic fibrosis (CF) is a complex, multi-system, life-shortening, autosomal recessive disease most common among Caucasians. Pulmonary pathology, the major cause of morbidity and mortality in CF, is characterized by dysregulation of cytokines and a vicious cycle of infection and inflammation. This cycle causes a progressive decline in lung function, eventually resulting in respiratory failure and death. The Th17 immune response plays an active role in the pathogenesis of CF pulmonary pathology, but it is not known whether the pathophysiology of CF disease contributes to a heightened Th17 response or whether CF naïve CD4+ T lymphocytes (Th0 cells) intrinsically have a heightened predisposition to Th17 differentiation. METHODS: To address this question, Th0 cells were isolated from the peripheral blood of CF mice, human CF subjects and corresponding controls. Murine Th0 cells were isolated from single spleen cell suspensions using fluorescence-activated cell sorting. Lymphocytes from human buffy coats were isolated by gradient centrifugation and Th0 cells were further isolated using a human naïve T cell isolation kit. Th0 cells were then assessed for their capacity to differentiate along Th17, Th1 or Treg lineages in response to corresponding cytokine stimulation. The T cell responses of human peripheral blood cells were also assessed ex vivo using flow cytometry. RESULTS: Here we identify in both mouse and human CF an intrinsically enhanced predisposition of Th0 cells to differentiate towards a Th17 phenotype, while having a normal propensity for differentiation into Th1 and Treg lineages. Furthermore, we identify an active Th17 response in the peripheral blood of human CF subjects. CONCLUSIONS: We propose that these novel observations offer an explanation, at least in part, for the known increased Th17-associated inflammation of CF and the early signs of inflammation in CF lungs before any evidence of infection. Moreover, these findings point towards direct modulation of T cell responses as a novel potential therapeutic strategy for combating excessive inflammation in CF.