Interactome Analysis of iPSC Secretome and Its Effect on Macrophages In Vitro.

Induced pluripotent stem cell secretome (iPSC-CM) mitigate organ injury and help in repair. Macrophages play a critical role in tissue repair and regeneration and can be directed to promote tissue repair by iPSC-CM, although the exact mechanisms are not known. In the current investigative study, we evaluated the possible mechanism by which iPSC-CM regulates the phenotype and secretory pattern of macrophages in vitro. Macrophages were obtained from human peripheral blood mononuclear cells and differentiated to various subpopulations and treated with either iPSC-CM or control media in vitro. Macrophage phenotype was assessed by flow cytometry, gene expression changes by qRT PCR and secretory pattern by multiplex protein analysis. The protein and gene interaction network revealed the involvement of Amyloid precursor protein (APP) and ELAV-like protein 1 (ELAVL-1) both present in the iPSC-CM to play an important role in regulating the macrophage phenotype and their secretory pattern. This exploratory study reveals, in part, the possible mechanism and identifies two potential targets by which iPSC-CM regulate macrophages and help in repair and regeneration.

In Vivo Electroporation-Mediated, Intrahepatic Alpha1 Antitrypsin Gene Transfer Reduces Pulmonary Emphysema in Pallid Mice.

RATIONALE: Mutation in the alpha1 antitrypsin (AAT) gene leads to low circulating levels of AAT, which is associated with several disease processes including pulmonary emphysema. The standard of care relies on substitution with plasma-purified AAT. We studied a novel approach to obtain sustained therapeutic levels of circulating AAT using nonviral in vivo electroporation-mediated gene transfer to the liver. METHODS: In vivo intrahepatic electroporation-mediated human AAT gene transfer was performed in C57 Bl/6J mice carrying a genetic deficiency of murine AAT (pallid mice) and suffering from pulmonary emphysema. The animals were evaluated for lung function using flexiVent and detailed stereological assessments. Lung neutrophilic burden was assessed. RESULTS: Pallid mice showed morphologically detectable pulmonary emphysema. Thirty days after in vivo electroporation-mediated gene transfer directly aimed at the liver, circulating human AAT was elevated and lung function was significantly improved compared to non-treated pallid mice. Stereological analysis revealed a reduction in pulmonary emphysema. CONCLUSION: Our data indicate that in vivo intrahepatic electroporation-mediated gene transfer of AAT is a safe and efficient procedure resulting in reduction of pulmonary emphysema in pallid mice.

Modulation of the unfolded protein response pathway as an antiviral approach in airway epithelial cells.

INTRODUCTION: Rhinovirus (RV) infection is a major cause of cystic fibrosis (CF) lung morbidity with limited therapeutic options. Various diseases involving chronic inflammatory response and infection are associated with endoplasmic reticulum (ER) stress and subsequent activation of the unfolded protein response (UPR), an adaptive response to maintain cellular homeostasis. Recent evidence suggests impaired ER stress response in CF airway epithelial cells, this might be a reason for recurrent viral infection in CF. Therefore, assuming that ER stress inducing drugs have antiviral properties, we evaluated the activation of the UPR by selected ER stress inducers as an approach to control virus replication in the CF bronchial epithelium. METHODS: We assessed the levels of UPR markers, namely the glucose-regulated protein 78 (Grp78) and the C/EBP homologous protein (CHOP), in primary CF and control bronchial epithelial cells and in a CF and control bronchial epithelial cell line before and after infection with RV. The cells were also pretreated with ER stress-inducing drugs and RV replication and shedding was measured by quantitative RT-PCR and by a TCID50 assay, respectively. Cell death was assessed by a lactate dehydrogenate (LDH) activity test in supernatants. RESULTS: We observed a significantly impaired induction of Grp78 and CHOP in CF compare to control cells following RV infection. The ER stress response could be significantly induced in CF cells by pharmacological ER stress inducers Brefeldin A, Tunicamycin, and Thapsigargin. The chemical induction of the UPR pathway prior to RV infection of CF and control cells reduced viral replication and shedding by up to two orders of magnitude and protected cells from RV-induced cell death. CONCLUSION: RV infection causes an impaired activation of the UPR in CF cells. Rescue of the ER stress response by chemical ER stress inducers reduced significantly RV replication in CF cells. Thus, pharmacological modulation of the UPR might represent a strategy to control respiratory virus replication in the CF bronchial epithelium.

Microbial and host immune factors as drivers of COPD.

Compartmentalisation of the respiratory tract microbiota in patients with different chronic obstructive pulmonary disease (COPD) severity degrees needs to be systematically investigated. In addition, it is unknown if the inflammatory and emphysematous milieux in patients with COPD are associated with changes in the respiratory tract microbiota and host macrophage gene expression. We performed a cross-sectional study to compare non-COPD controls (n=10) to COPD patients (n=32) with different disease severity degrees. Samples (n=187) were obtained from different sites of the upper and lower respiratory tract. Microbiota analyses were performed by 16S ribosomal RNA gene sequencing and host gene expression analyses by quantitative real-time PCR of distinct markers of bronchoalveolar lavage cells. Overall, the microbial communities of severe COPD (Global Initiative for Chronic Obstructive Lung Disease (GOLD) grade 3/4) patients clustered significantly differently to controls and less severe COPD (GOLD 1/2) patients (permutational multivariate ANOVA (MANOVA), p=0.001). However, we could not detect significant associations between the different sampling sites in the lower airways. In addition, the chosen set of host gene expression markers significantly separated COPD GOLD 3/4 patients, and we found correlations between the composition of the microbiota and the host data. In conclusion, this study demonstrates associations between host gene expression and microbiota profiles that may influence the course of COPD.

Hepatocyte growth factor secreted by bone marrow stem cell reduce ER stress and improves repair in alveolar epithelial II cells.

Idiopathic Pulmonary Fibrosis (IPF) is a progressive, irreversible lung disease with complex pathophysiology. Evidence of endoplasmic reticulum (ER) stress has been reported in alveolar epithelial cells (AEC) in IPF patients. Secreted mediators from bone marrow stem cells (BMSC-cm) have regenerative properties. In this study we investigate the beneficial effects of BMSC-cm on ER stress response in primary AEC and ER stressed A549 cells. We hypothesize that BMSC-cm reduces ER stress. Primary AEC isolated from IPF patients were treated with BMSC-cm. To induce ER stress A549 cells were incubated with Tunicamycin or Thapsigargin and treated with BMSC-cm, or control media. Primary IPF-AEC had high Grp78 and CHOP gene expression, which was lowered after BMSC-cm treatment. Similar results were observed in ER stressed A549 cells. Alveolar epithelial repair increased in presence of BMSC-cm in ER stressed A549 cells. Hepatocyte growth factor (HGF) was detected in biologically relevant levels in BMSC-cm. Neutralization of HGF in BMSC-cm attenuated the beneficial effects of BMSC-cm including synthesis of surfactant protein C (SP-C) in primary AEC, indicating a crucial role of HGF in ER homeostasis and alveolar epithelial repair. Our data suggest that BMSC-cm may be a potential therapeutic option for treating pulmonary fibrosis.

Mobility and shape adaptation of neutrophil in the microchannel flow.

This paper presents motion of neutrophil in a confined environment. Many experimental and theoretical studies were performed to show mechanics and basic principles of the white blood cell motion. However, they were mostly performed on flat plates without boundaries. More realistic model of flow in the capillaries based on confinement, curvature and adequate dimensions is applied in our experiments. These conditions lead to cell motion with deformability and three-dimensional character of that movement. Neutrophils are important cells for human immune system. Their motion and attachment often influence several diseases and immune response. Hence, studies focus on that particular cell type. We have shown that deformability of the cell influences its velocity. Cells actively participate in the flow using the shear gradient to advance control motion. The observed neutrophil velocity was from 1 up to 100µm/s.

Renal tubular SGK1 deficiency causes impaired K+ excretion via loss of regulation of NEDD4-2/WNK1 and ENaC.

The stimulation of postprandial K(+) clearance involves aldosterone-independent and -dependent mechanisms. In this context, serum- and glucocorticoid-induced kinase (SGK)1, a ubiquitously expressed kinase, is one of the primary aldosterone-induced proteins in the aldosterone-sensitive distal nephron. Germline inactivation of SGK1 suggests that this kinase is fundamental for K(+) excretion under conditions of K(+) load, but the specific role of renal SGK1 remains elusive. To avoid compensatory mechanisms that may occur during nephrogenesis, we used inducible, nephron-specific Sgk1(Pax8/LC1) mice to assess the role of renal tubular SGK1 in K(+) regulation. Under a standard diet, these animals exhibited normal K(+) handling. When challenged by a high-K(+) diet, they developed severe hyperkalemia accompanied by a defect in K(+) excretion. Molecular analysis revealed reduced neural precursor cell expressed developmentally downregulated protein (NEDD)4-2 phosphorylation and total expression. γ-Epithelial Na(+) channel (ENaC) expression and α/γENaC proteolytic processing were also decreased in mutant mice. Moreover, with no lysine kinase (WNK)1, which displayed in control mice punctuate staining in the distal convoluted tubule and diffuse distribution in the connecting tubule/cortical colleting duct, was diffused in the distal convoluted tubule and less expressed in the connecting tubule/collecting duct of Sgk(Pax8/LC1) mice. Moreover, Ste20-related proline/alanine-rich kinase phosphorylation, and Na(+)-Cl(-) cotransporter phosphorylation/apical localization were reduced in mutant mice. Consistent with the altered WNK1 expression, increased renal outer medullary K(+) channel apical localization was observed. In conclusion, our data suggest that renal tubular SGK1 is important in the regulation of K(+) excretion via the control of NEDD4-2, WNK1, and ENaC.

Size-dependent accumulation of particles in lysosomes modulates dendritic cell function through impaired antigen degradation.

INTRODUCTION: Nanosized particles may enable therapeutic modulation of immune responses by targeting dendritic cell (DC) networks in accessible organs such as the lung. To date, however, the effects of nanoparticles on DC function and downstream immune responses remain poorly understood. METHODS: Bone marrow-derived DCs (BMDCs) were exposed in vitro to 20 or 1,000 nm polystyrene (PS) particles. Particle uptake kinetics, cell surface marker expression, soluble protein antigen uptake and degradation, as well as in vitro CD4(+) T-cell proliferation and cytokine production were analyzed by flow cytometry. In addition, co-localization of particles within the lysosomal compartment, lysosomal permeability, and endoplasmic reticulum stress were analyzed. RESULTS: The frequency of PS particle-positive CD11c(+)/CD11b(+) BMDCs reached an early plateau after 20 minutes and was significantly higher for 20 nm than for 1,000 nm PS particles at all time-points analyzed. PS particles did not alter cell viability or modify expression of the surface markers CD11b, CD11c, MHC class II, CD40, and CD86. Although particle exposure did not modulate antigen uptake, 20 nm PS particles decreased the capacity of BMDCs to degrade soluble antigen, without affecting their ability to induce antigen-specific CD4(+) T-cell proliferation. Co-localization studies between PS particles and lysosomes using laser scanning confocal microscopy detected a significantly higher frequency of co-localized 20 nm particles as compared with their 1,000 nm counterparts. Neither size of PS particle caused lysosomal leakage, expression of endoplasmic reticulum stress gene markers, or changes in cytokines profiles. CONCLUSION: These data indicate that although supposedly inert PS nanoparticles did not induce DC activation or alteration in CD4(+) T-cell stimulating capacity, 20 nm (but not 1,000 nm) PS particles may reduce antigen degradation through interference in the lysosomal compartment. These findings emphasize the importance of performing in-depth analysis of DC function when developing novel approaches for immune modulation with nanoparticles.

Molecular characterization of two novel cases of complete complement inhibitor Factor I deficiency.

Factor I (FI) is the major complement inhibitor that degrades activated complement components C3b and C4b in the presence of specific cofactors. Complete FI deficiency results in secondary complement deficiency due to uncontrolled spontaneous alternative pathway activation. In this study we describe two unrelated patients with complete FI deficiency and undetectable alternative complement pathway activity. Both patients had experienced recurrent infections and arthralgia/arthritis. In one patient, analysis of genomic DNA revealed deletion of two adenine nucleotides in exon 2 of the CFI gene (c.133-134delAA), causing a frame shift and premature STOP codon/termination in the FIMAC (FI-membrane attack complex) domain (p.K45SfsX11). The other patient carried an A>T substitution in exon 6 (c.866A>T) encoding the LDLr2 (low density lipoprotein receptor) domain (p.D289V), resulting in an aspartic acid to valine change. Both patients were homozygous for the mutations while their healthy parents were heterozygous carriers. The mutations were introduced into recombinant FI, causing lack of FI expression and secretion upon transient transfection. Mutation p.K45SfsX11 theoretically allows expression of a 55 amino acid fragment of FI that lacks the serine protease domain, preventing proteolytic activity. In contrast, aspartic acid D289 is crucial for folding of FI. This report describes the molecular and functional consequences of two novel mutations of FI, providing a unique insight into the pathogenesis of complete FI deficiency in these patients.

Analysis of binding sites on complement factor I that are required for its activity.

The central complement inhibitor factor I (FI) degrades activated complement factors C4b and C3b in the presence of cofactors such as C4b-binding protein, factor H, complement receptor 1, and membrane cofactor protein. FI is a serine protease composed of two chains. The light chain comprises the serine protease domain, whereas the heavy chain contains several domains; that is, the FI and membrane attack complex domain (FIMAC), CD5, low density lipoprotein receptor 1 (LDLr1) and LDLr2 domains. To understand better how FI acts as a complement inhibitor, we used homology-based models of FI domains to predict potential binding sites. Specific amino acids were then mutated to yield 16 well expressed mutants, which were then purified from media of eukaryotic cells for functional analyses. The Michaelis constant (K(m)) of all FI mutants toward a small substrate was not altered, whereas some mutants showed increased maximum initial velocity (V(max)). All the mutations in the FIMAC domain affected the ability of FI to degrade C4b and C3b irrespective of the cofactor used, whereas only some mutations in the CD5 and LDLr1/2 domains had a similar effect. These same mutants also showed impaired binding to C3met. In conclusion, the FIMAC domain appears to harbor the main binding sites important for the ability of FI to degrade C4b and C3b.

Low plasma levels of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-alpha (TNFalpha), and vascular endothelial growth factor (VEGF) in patients with alpha1-antitrypsin deficiency-related fibromyalgia.

Abnormalities in blood inflammatory markers have been associated with clinical manifestations and the pathogenesis of the fibromyalgia syndrome (FMS); a relationship between inherited alpha1-antitrypsin deficiency (AATD) and FMS has also been recently raised. In this study, plasma levels of inflammatory markers in FMS patients with and without AATD have been investigated. Blood samples from 138 age-matched females (79 FMS) and 59 general population (GP), with normal MM [n = 82 (59.4%)] and with MS, MZ, SZ, and ZZ AATD genotypes [n = 56 (40.6%)], were analyzed by ELISA for monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-alpha (TNFalpha), soluble TNFalpha receptors I and II, interleukin-8, and vascular endothelial growth factor (VEGF). Plasma levels of MCP-1, VEGF, and TNFalpha were significantly lower in FMS and GP subjects with AATD compared with those with normal MM-AAT genotypes. Moreover, plasma levels of MCP-1, VEGF, and TNFalpha were lower in AATD subjects with FMS than in those without FMS (P = 0.000, 0.000, and 0.046, respectively). No statistical differences were found for the other substances measured. Furthermore, a logistic regression model based on plasma MCP-1 cutoff value of

Alpha1-antitrypsin inhibits the activity of the matriptase catalytic domain in vitro.

Matriptase is a type II transmembrane protease that is characterized by an N-terminal transmembrane and multiple extracellular domains, in addition to the conserved extracellular serine protease catalytic domain. The expression pattern of matriptase suggests that this protease may play broad roles in the biology of surface lining epithelial cells. In this study we report that alpha1-antitrypsin (AAT), an endogenous inhibitor of serine proteases, inhibits the catalytic domain of human recombinant matriptase in vitro. Co-incubation of AAT with matriptase (at a molar ratio 1:2) resulted in the formation of heat stable complexes, clearly seen in sodium dodecyl sulfate electrophoresis and Western blots. AAT was found to be a slow, tight-binding inhibitor of the catalytic domain of matriptase with a second order reaction rate constant of 0.31 x 10(3) M(-1)s(-1). Notably, the oxidized form of AAT, which lacks serine protease inhibitor activity, failed to generate matriptase complexes and to inhibit matriptase activity. Since matriptase is involved in a number of physiologic processes, including activation of epithelial sodium channels, our findings offer considerable new insights into new regulatory function of AAT in vivo.

alpha1-Antitrypsin regulates CD14 expression and soluble CD14 levels in human monocytes in vitro.

The recognition of bacterial lipopolysaccharide (LPS) is principally mediated by either membrane-bound or soluble form of the glycoprotein CD14 and CD14-associated signal transducer, toll-like receptor 4 (TLR4). Recent findings indicate that the serine protease inhibitor, alpha1-antitrypsin (AAT), may not only afford protection against proteolytic injury, but may also neutralize microbial activities and affect regulation of innate immunity. We postulated that AAT affects monocyte responses to LPS by regulating CD14 expression and soluble CD14 release. Here we show that a short-term (up to 2h) monocyte exposure to AAT alone or in combination with LPS leads to a remarkable induction of CD14 levels. In parallel, a short-term (2h) cell exposure to AAT/LPS significantly enhances LPS-induced NF kappaB (p50 and p65) activation in conjunction with increased TNFalpha, IL-1 beta and IL-8 release. In contrast, longer term incubation (18 h) of monocytes with combined AAT/LPS results in a significant reduction in expression of both CD14 and TLR4, inhibition of LPS-induced TNFalpha, IL-1 beta and IL-8 mRNA and protein expression. These findings provide evidence that AAT is an important regulator of CD14 expression and release in monocytes and suggest that AAT may be involved in LPS neutralization and prevention of over-activation of monocytes in vivo.

Alpha1-antitrypsin, old dog, new tricks. Alpha1-antitrypsin exerts in vitro anti-inflammatory activity in human monocytes by elevating cAMP.

Regulation of serine protease activity is considered to be the sole mechanism for the function of alpha1-antitrypsin (AAT). However, recent reports of the anti-inflammatory effects of AAT are hard to reconcile with this classical mechanism. We discovered that two key activities of AAT in vitro, namely inhibition of endotoxin-stimulated tumor necrosis factor-alpha and enhancement of interleukin-10 in human monocytes, are mediated by an elevation of cAMP and activation of cAMP-dependent protein kinase A. As expected with this type of mechanism, the AAT-mediated rise in cAMP and the impact on endotoxin-stimulated tumor necrosis factor-alpha and interleukin-10 was enhanced when the catabolism of cAMP was blocked by the phosphodiesterase inhibitor rolipram. These effects were still observed with modified forms of AAT lacking protease inhibitor activity.

Alpha1-antitrypsin inhibits Moraxella catarrhalis MID protein-induced tonsillar B cell proliferation and IL-6 release.

Alpha1-antitrypsin (AAT) is a major circulating and tissues inhibitor of serine proteinases implicated in the regulation of inflammation and host defence. There is now increasing evidence that AAT may also exhibit anti-inflammatory activities independent of its protease inhibitor function. This study was undertaken to investigate the effects of native (inhibitory) and polymerized (non-inhibitory) forms of AAT on MID (Moraxella IgD binding protein)-induced human tonsillar B cell activation in vitro. We found that 0.5 microg/ml MID induces B cell proliferation and stimulates IL-6 release (p<0.001) relative to non-stimulated controls. Both native and polymerized AAT (0.5 mg/ml) inhibited MID-stimulated B cell proliferation in a similar manner (by 70%, p<0.001), whereas MID-induced IL-6 release was more strongly suppressed by polymerized (9.9-fold, p<0.001) as compared to native AAT (2.8-fold, p<0.01). Electrophoretic analysis of cell culture media did not indicate any interaction between AAT and MID, and flow cytometry data showed no competition for the same receptor. The effects of AATs were observed whether added together with MID or 2h after MID-addition to cell cultures. Thus, our data demonstrate that AAT inhibits MID-induced B cell activation in vitro that is unrelated to its protease inhibitory activity and is not dependent on MID binding to the cell surface.

Prolastin, a pharmaceutical preparation of purified human alpha1-antitrypsin, blocks endotoxin-mediated cytokine release.

BACKGROUND: Alpha1-antitrypsin (AAT) serves primarily as an inhibitor of the elastin degrading proteases, neutrophil elastase and proteinase 3. There is ample clinical evidence that inherited severe AAT deficiency predisposes to chronic obstructive pulmonary disease. Augmentation therapy for AAT deficiency has been available for many years, but to date no sufficient data exist to demonstrate its efficacy. There is increasing evidence that AAT is able to exert effects other than protease inhibition. We investigated whether Prolastin, a preparation of purified pooled human AAT used for augmentation therapy, exhibits anti-bacterial effects. METHODS: Human monocytes and neutrophils were isolated from buffy coats or whole peripheral blood by the Ficoll-Hypaque procedure. Cells were stimulated with lipopolysaccharide (LPS) or zymosan, either alone or in combination with Prolastin, native AAT or polymerised AAT for 18 h, and analysed to determine the release of TNFalpha, IL-1beta and IL-8. At 2-week intervals, seven subjects were submitted to a nasal challenge with sterile saline, LPS (25 microg) and LPS-Prolastin combination. The concentration of IL-8 was analysed in nasal lavages performed before, and 2, 6 and 24 h after the challenge. RESULTS: In vitro, Prolastin showed a concentration-dependent (0.5 to 16 mg/ml) inhibition of endotoxin-stimulated TNFalpha and IL-1beta release from monocytes and IL-8 release from neutrophils. At 8 and 16 mg/ml the inhibitory effects of Prolastin appeared to be maximal for neutrophil IL-8 release (5.3-fold, p < 0.001 compared to zymosan treated cells) and monocyte TNFalpha and IL-1beta release (10.7- and 7.3-fold, p < 0.001, respectively, compared to LPS treated cells). Furthermore, Prolastin (2.5 mg per nostril) significantly inhibited nasal IL-8 release in response to pure LPS challenge. CONCLUSION: Our data demonstrate for the first time that Prolastin inhibits bacterial endotoxin-induced pro-inflammatory responses in vitro and in vivo, and provide scientific bases to explore new Prolastin-based therapies for individuals with inherited AAT deficiency, but also for other clinical conditions.