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1.  Identification of a Role for the PI3K/AKT/mTOR Signaling Pathway in Innate Immune Cells 
PLoS ONE  2014;9(4):e94496.
The innate immune system is the first line of host defense against infection and involves several different cell types. Here we investigated the role of the phosphatidylinositol 3 kinase (PI3K) signaling pathway in innate immune cells. By blocking this pathway with pharmacological inhibitors, we found that the production of proinflammatory cytokines was drastically suppressed in monocytes and macrophages. Further study revealed that the suppression was mainly related to the mammalian target of rapamycin (mTOR)/p70S6K signaling. In addition, we found that the PI3K pathway was involved in macrophage motility and neovascularization. Our data provide a rationale that inhibition of the PI3K signaling pathway could be an attractive approach for the management of inflammatory disorders.
doi:10.1371/journal.pone.0094496
PMCID: PMC3981814  PMID: 24718556
2.  Immune Dysregulation as a Cause of Autoinflammation in Fragile X Premutation Carriers: Link between FMRI CGG Repeat Number and Decreased Cytokine Responses 
PLoS ONE  2014;9(4):e94475.
Background
Increased rates of autoinflammatory and autoimmune disorders have been observed in female premutation carriers of CGG repeat expansion alleles of between 55–200 repeats in the fragile X mental retardation 1 (FMR1) gene. To determine whether an abnormal immune profile was present at a cellular level that may predispose female carriers to autoinflammatory conditions, we investigated dynamic cytokine production following stimulation of blood cells. In addition, splenocyte responses were examined in an FMR1 CGG knock-in mouse model of the fragile X premutation.
Methods
Human monocyte and peripheral blood leukocytes (PBLs) were isolated from the blood of 36 female FMR1 premutation carriers and 15 age-matched controls. Cells were cultured with media alone, LPS or PHA. In the animal model, splenocytes were isolated from 32 CGG knock-in mice and 32 wild type littermates. Splenocytes were cultured with media alone or LPS or PMA/Ionomycin. Concentrations of cytokines (GM-CSF, IL-1β, IL-6, IL-10, IL-13, IL-17, IFNγ, TNFα, and MCP-1) were determined from the supernatants of cellular cultures via Luminex multiplex assay. Additionally, phenotypic cellular markers were assessed on cells isolated from human subjects via flow cytometry.
Results
We found decreases in cytokine production in human premutation carriers as well as in the FMR1 knock-in mice when compared with controls. Levels of cytokines were found to be associated with CGG repeat length in both human and mouse. Furthermore, T cells from human premutation carriers showed decreases in cell surface markers of activation when compared with controls.
Conclusions
In this study, FMR1 CGG repeat expansions are associated with decreased immune responses and immune dysregulation in both humans and mice. Deficits in immune responses in female premutation carriers may lead to increased susceptibility to autoimmunity and further research is warranted to determine the link between FMR1 CGG repeat lengths and onset of autoinflammatory conditions.
doi:10.1371/journal.pone.0094475
PMCID: PMC3981824  PMID: 24718368
3.  Routes of Allergic Sensitization and Myeloid Cell IKKβ Differentially Regulate Antibody Responses and Allergic Airway Inflammation in Male and Female Mice 
PLoS ONE  2014;9(3):e92307.
Gender influences the incidence and/or the severity of several diseases and evidence suggests a higher rate of allergy and asthma among women. Most experimental models of allergy use mice sensitized via the parenteral route despite the fact that the mucosal tissues of the gastrointestinal and respiratory tracts are major sites of allergic sensitization and/or allergic responses. We analyzed allergen-specific Ab responses in mice sensitized either by gavage or intraperitoneal injection of ovalbumin together with cholera toxin as adjuvant, as well as allergic inflammation and lung functions following subsequent nasal challenge with the allergen. Female mice sensitized intraperitoneally exhibited higher levels of serum IgE than their male counterparts. After nasal allergen challenge, these female mice expressed higher Th2 responses and associated inflammation in the lung than males. On the other hand, male and female mice sensitized orally developed the same levels of allergen-specific Ab responses and similar levels of lung inflammation after allergen challenge. Interestingly, the difference in allergen-specific Ab responses between male and female mice sensitized by the intraperitoneal route was abolished in IKKβΔMye mice, which lack IKKβ in myeloid cells. In summary, the oral or systemic route of allergic sensitization and IKKβ signaling in myeloid cells regulate how the gender influences allergen-specific responses and lung allergic inflammation.
doi:10.1371/journal.pone.0092307
PMCID: PMC3965427  PMID: 24667561
4.  Progranulin-Derived Atsttrin Directly Binds to TNFRSF25 (DR3) and Inhibits TNF-Like Ligand 1A (TL1A) Activity 
PLoS ONE  2014;9(3):e92743.
Atsttrin, a progranulin (PGRN)-derived molecule composed of three TNFR-binding domains of PGRN, binds to TNF receptors (TNFR) and is therapeutic against inflammatory arthritis. Here we screened the associations of Atsttrin and other members in TNFR subfamily, which led to the discovery of TNFRSF25 (DR3) as an additional Atsttrin-interacting member in TNFR family. Similar to TNFR1 and TNFR2, DR3 also directly bound to Atsttrin. The first three cysteine-rich domains (CRD) in the extracellular portion of DR3 were required for this interaction. Atsttrin inhibited the interaction between DR3 and its TNF-Like Ligand 1A (TL1A). In addition, Atsttrin inhibited TL1A-stimulated target gene expressions and neutralized TL1A-enhanced osteoclastogenesis in vitro. Furthermore, Atsttrin ameliorated the pathology in dextran sulfate sodium induced colitis. Taken together, these findings not only provide the new insights into Atsttrin's therapeutic action in inflammatory arthritis, but may also present Atsttrin as a novel biological agent for treating various types of diseases associated with TL1A/DR3 pathway.
doi:10.1371/journal.pone.0092743
PMCID: PMC3961393  PMID: 24651300
5.  Viral Replication and Lung Lesions in BALB/c Mice Experimentally Inoculated with Avian Metapneumovirus Subgroup C Isolated from Chickens 
PLoS ONE  2014;9(3):e92136.
Avian metapneumovirus (aMPV) emerged as an important respiratory pathogen causing acute respiratory tract infection in avian species. Here we used a chicken aMPV subgroup C (aMPV/C) isolate to inoculate experimentally BALB/c mice and found that the aMPV/C can efficiently replicate and persist in the lungs of mice for at least 21 days with a peak viral load at day 6 postinoculation. Lung pathological changes were characterized by increased inflammatory cells. Immunochemical assay showed the presence of viral antigens in the lungs and significant upregulation of pulmonary inflammatory cytokines and chemokines including MCP-1, MIP-1α, RANTES, IL-1β, IFN-γ, and TNF-α were detected following inoculation. These results indicate for the first time that chicken aMPV/C may replicate in the lung of mice. Whether aMPV/C has potential as zoonotic pathogen, further investigation will be required.
doi:10.1371/journal.pone.0092136
PMCID: PMC3956885  PMID: 24637582
6.  Probiotics Protect Mice from Ovariectomy-Induced Cortical Bone Loss 
PLoS ONE  2014;9(3):e92368.
The gut microbiota (GM) modulates the hosts metabolism and immune system. Probiotic bacteria are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host and can alter the composition of the GM. Germ-free mice have increased bone mass associated with reduced bone resorption indicating that the GM also regulates bone mass. Ovariectomy (ovx) results in bone loss associated with altered immune status. The purpose of this study was to determine if probiotic treatment protects mice from ovx-induced bone loss. Mice were treated with either a single Lactobacillus (L) strain, L. paracasei DSM13434 (L. para) or a mixture of three strains, L. paracasei DSM13434, L. plantarum DSM 15312 and DSM 15313 (L. mix) given in the drinking water during 6 weeks, starting two weeks before ovx. Both the L. para and the L. mix treatment protected mice from ovx-induced cortical bone loss and bone resorption. Cortical bone mineral content was higher in both L. para and L. mix treated ovx mice compared to vehicle (veh) treated ovx mice. Serum levels of the resorption marker C-terminal telopeptides and the urinary fractional excretion of calcium were increased by ovx in the veh treated but not in the L. para or the L. mix treated mice. Probiotic treatment reduced the expression of the two inflammatory cytokines, TNFα and IL-1β, and increased the expression of OPG, a potent inhibitor of osteoclastogenesis, in cortical bone of ovx mice. In addition, ovx decreased the frequency of regulatory T cells in bone marrow of veh treated but not probiotic treated mice. In conclusion, treatment with L. para or the L. mix prevents ovx-induced cortical bone loss. Our findings indicate that these probiotic treatments alter the immune status in bone resulting in attenuated bone resorption in ovx mice.
doi:10.1371/journal.pone.0092368
PMCID: PMC3956931  PMID: 24637895
7.  Development of a Novel Severe Triple Allergen Asthma Model in Mice Which Is Resistant to Dexamethasone and Partially Resistant to TLR7 and TLR9 Agonist Treatment 
PLoS ONE  2014;9(3):e91223.
Severe asthma is characterised by persistent inflammation, hyperreactivity and remodeling of the airways. No efficient treatment is available, this is particularly the case for steroid resistant phenotypes. Our aim therefore was to develop a preclinical model showing characteristics of severe human asthma including steroid insensitivity. Mice were first sensitized with ovalbumin, extracts of cockroach or house dust mite followed by a challenge period of seven weeks. Further to this, an additional group of mice was sensitized with all three allergens and then challenged with allergen alternating weekly between allergens. All three allergens applied separately to the mice induced comparably strong Th2-type airway inflammation, airway hyperreactivity and airway remodeling, which was characterised by fibrosis and increased smooth muscle thickness. In contrast, application of all three allergens together resulted in a greater Th2 response and increased airway hyperreactivity and a stronger albeit not significant remodeling phenotype compared to using HDM or CRA. In this triple allergen model dexamethasone application, during the last 4 weeks of challenge, showed no suppressive effects on any of these parameters in this model. In contrast, both TLR7 agonist resiquimod and TLR9 agonist CpG-ODN reduced allergen-specific IgE, eosinophils, and collagen I in the lungs. The TLR9 agonist also reduced IL-4 and IL-5 whilst increasing IFN-γ and strongly IL-10 levels in the lungs, effects not seen with the TLR7 agonist. However, neither TLR agonist had any effect on airway hyperreactivity and airway smooth muscle mass. In conclusion we have developed a severe asthma model, which is steroid resistant and only partially sensitive to TLR7 and TLR9 agonist treatment. This model may be particular useful to test new potential therapeutics aiming at treating steroid resistant asthma in humans and investigating the underlying mechanisms responsible for steroid insensitivity.
doi:10.1371/journal.pone.0091223
PMCID: PMC3949744  PMID: 24618687
8.  Collective nitric oxide production provides tissue-wide immunity during Leishmania infection  
The Journal of Clinical Investigation  2014;124(4):1711-1722.
Nitric oxide (NO) production is critical for the host defense against intracellular pathogens; however, it is unclear whether NO-dependent control of intracellular organisms depends on cell-intrinsic or cell-extrinsic activity of NO. For example, NO production by infected phagocytes may enable these cells to individually control their pathogen burden. Alternatively, the ability of NO to diffuse across cell membranes might be critical for infection control. Here, using a murine ear infection model, we found that, during infection with the intracellular parasite Leishmania major, expression of inducible NO synthase does not confer a cell-intrinsic ability to lower parasite content. We demonstrated that the diffusion of NO promotes equally effective parasite killing in NO-producing and bystander cells. Importantly, the collective production of NO by numerous phagocytes was necessary to reach an effective antimicrobial activity. We propose that, in contrast to a cell-autonomous mode of pathogen control, this cooperative mechanism generates an antimicrobial milieu that provides the basis for pathogen containment at the tissue level.
doi:10.1172/JCI72058
PMCID: PMC3973105  PMID: 24614106
9.  Formylpeptide Receptors Mediate Rapid Neutrophil Mobilization to Accelerate Wound Healing 
PLoS ONE  2014;9(3):e90613.
Wound healing is a multi-phased pathophysiological process requiring chemoattractant receptor-dependent accumulation of myeloid cells in the lesion. Two G protein-coupled formylpeptide receptors Fpr1 and Fpr2 mediate rapid neutrophil infiltration in the liver of Listeria-infected mice by sensing pathogen-derived chemotactic ligands. These receptors also recognize host-derived chemotactic peptides in inflammation and injury. Here we report the capacity of Fprs to promote the healing of sterile skin wound in mice by initiating neutrophil infiltration. We found that in normal miceneutrophils rapidly infiltrated the dermis in the wound before the production of neutrophil-specific chemokines by the injured tissue. In contrast, rapid neutrophil infiltration was markedly reduced with delayed wound closure in mice deficient in both Fprs. In addition, we detected Fpr ligand activity that chemoattracted neutrophils into the wound tissue. Our study thus demonstrates that Fprs are critical for normal healing of the sterile skin wound by mediating the first wave of neutrophil infiltration.
doi:10.1371/journal.pone.0090613
PMCID: PMC3946181  PMID: 24603667
10.  TWEAK Promotes Peritoneal Inflammation 
PLoS ONE  2014;9(3):e90399.
Peritoneal dialysis (PD) is complicated by peritonitis episodes that cause loss of mesothelium and eventually sclerosing peritonitis. An improved understanding of the molecular contributors to peritoneal injury and defense may increase the therapeutic armamentarium to optimize peritoneal defenses while minimizing peritoneal injury. There is no information on the expression and function of the cytokine TWEAK and its receptor Fn14 during peritoneal injury. Fn14 expression and soluble TWEAK levels were measured in human PD peritoneal effluent cells or fluids with or without peritonitis. Fn14 expression was also analyzed in peritoneal biopsies from PD patients. Actions of intraperitoneal TWEAK were studied in mice in vivo. sTWEAK levels were increased in peritoneal effluent in PD peritonitis. Effluent sTWEAK levels correlated with the number of peritoneal macrophages (r = 0.491, p = 0.002). Potential TWEAK targets that express the receptor Fn14 include mesothelial cells and macrophages, as demonstrated by flow cytometry of peritoneal effluents and by analysis of peritoneal biopsies. Peritoneal biopsy Fn14 correlated with mesothelial injury, fibrosis and inflammation, suggesting a potential deleterious effect of TWEAK/Fn14. In this regard, intraperitoneal TWEAK administration to mice promoted peritoneal inflammation characterized by increased peritoneal effluent MCP-1, Fn14 and Gr1+ macrophages, increased mesothelial Fn14, MCP-1 and CCL21 expression and submesothelial tissue macrophage recruitment. Taken together these data suggest that the TWEAK/Fn14 system may promote inflammation and tissue injury during peritonitis and PD.
doi:10.1371/journal.pone.0090399
PMCID: PMC3944020  PMID: 24599047
11.  Anti-Inflammatory and Anti-Remodelling Effects of ISU201, a Modified Form of the Extracellular Domain of Human BST2, in Experimental Models of Asthma: Association with Inhibition of Histone Acetylation 
PLoS ONE  2014;9(3):e90436.
There are few alternatives to glucocorticosteroids for treatment of asthma. We assessed the activity of a novel protein drug designated ISU201, the extracellular domain of the human cell surface protein BST2, stabilised by fusion with the Fc region of IgG, in mouse models of mild chronic asthma and an acute exacerbation of asthma. The ability of ISU201 to suppress airway inflammation and remodelling was compared with that of dexamethasone. Female BALB/c mice were systemically sensitised with ovalbumin, then received controlled low-level challenge with aerosolised ovalbumin for 6 weeks, which induced lesions of mild chronic asthma, and were treated with drugs during the final 2 weeks. Alternatively, sensitised mice received 4 weeks of chronic low-level challenge and were treated 24 and 2 hours before a final single moderate-level challenge, which triggered acute airway inflammation simulating an asthmatic exacerbation. Inflammation and remodelling were quantified, as was the expression of pro-inflammatory cytokines in bronchoalveolar lavage fluid and tissues. To identify cellular targets of ISU201, we assessed the effects of the drug on activated lymphocytes, macrophages and airway epithelial cells. In the model of mild chronic asthma, ISU201 was as effective as dexamethasone in suppressing airway inflammation and most changes of remodelling. In the model of an allergen-induced acute exacerbation of chronic asthma, ISU201 was also an effective anti-inflammatory agent, although it was less active than dexamethasone. The drug acted on multiple cellular targets, suppressing production of pro-inflammatory cytokines by lymphocytes and macrophages. ISU201 significantly reduced acetylation of histone H4 in airway epithelial cells, suggesting at least one potential mechanism of action. We conclude that in these models of asthma, ISU201 is a broad-spectrum inhibitor of both airway inflammation and remodelling. Thus, unlike drugs which target specific mediators, it could potentially be an alternative or an adjunct to glucocorticoids for the treatment of asthma.
doi:10.1371/journal.pone.0090436
PMCID: PMC3940910  PMID: 24594933
12.  Ciprofloxacin Enhances Stress Erythropoiesis in Spleen and Increases Survival after Whole-Body Irradiation Combined with Skin-Wound Trauma 
PLoS ONE  2014;9(2):e90448.
Severe hematopoietic loss is one of the major therapeutic targets after radiation-combined injury (CI), a kind of injury resulting from radiation exposure combined with other traumas. In this study, we tested the use of ciprofloxacin (CIP) as a treatment, because of recently reported immunomodulatory effects against CI that may improve hematopoiesis. The CIP regimen was a daily, oral dose for 3 weeks, with the first dose 2 h after CI. CIP treatment improved 30-day survival in mice at 80% compared to 35% for untreated controls. Study of early changes in hematological parameters identified CI-induced progressive anemia by 10 days that CIP significantly ameliorated. CI induced erythropoietin (EPO) mRNA in kidney and protein in kidney and serum; CIP stimulated EPO mRNA expression. In spleens of CI mice, CIP induced bone morphogenetic protein 4 (BMP4) in macrophages with EPO receptors. Splenocytes from CIP-treated CI mice formed CD71+ colony-forming unit-erythroid significantly better than those from controls. Thus, CIP-mediated BMP4-dependent stress erythropoiesis may play a role in improving survival after CI.
doi:10.1371/journal.pone.0090448
PMCID: PMC3938753  PMID: 24587369
13.  Soluble TNFRp75 regulates host protective immunity against Mycobacterium tuberculosis 
The Journal of Clinical Investigation  2014;124(4):1537-1551.
Development of host protective immunity against Mycobacterium tuberculosis infection is critically dependent on the inflammatory cytokine TNF. TNF signals through 2 receptors, TNFRp55 and TNFRp75; however, the role of TNFRp75-dependent signaling in immune regulation is poorly defined. Here we found that mice lacking TNFRp75 exhibit greater control of M. tuberculosis infection compared with WT mice. TNFRp75–/– mice developed effective bactericidal granulomas and demonstrated increased pulmonary recruitment of activated DCs. Moreover, IL-12p40–dependent migration of DCs to lung draining LNs of infected TNFRp75–/– mice was substantially higher than that observed in WT M. tuberculosis–infected animals and was associated with enhanced frequencies of activated M. tuberculosis–specific IFN-γ–expressing CD4+ T cells. In WT mice, TNFRp75 shedding correlated with markedly reduced bioactive TNF levels and IL-12p40 expression. Neutralization of TNFRp75 in M. tuberculosis–infected WT BM-derived DCs (BMDCs) increased production of bioactive TNF and IL-12p40 to a level equivalent to that produced by TNFRp75–/– BMDCs. Addition of exogenous TNFRp75 to TNFRp75–/– BMDCs infected with M. tuberculosis decreased IL-12p40 synthesis, demonstrating that TNFRp75 shedding regulates DC activation. These data indicate that TNFRp75 shedding downmodulates protective immune function and reduces host resistance and survival; therefore, targeting TNFRp75 may be beneficial for improving disease outcome.
doi:10.1172/JCI45005
PMCID: PMC3973106  PMID: 24569452
14.  Interleukin-33 Increases Antibacterial Defense by Activation of Inducible Nitric Oxide Synthase in Skin 
PLoS Pathogens  2014;10(2):e1003918.
Interleukin-33 (IL-33) is associated with multiple diseases, including asthma, rheumatoid arthritis, tissue injuries and infections. Although IL-33 has been indicated to be involved in Staphylococcus aureus (S. aureus) wound infection, little is known about how IL-33 is regulated as a mechanism to increase host defense against skin bacterial infections. To explore the underlying intricate mechanism we first evaluated the expression of IL-33 in skin from S. aureus-infected human patients. Compared to normal controls, IL-33 was abundantly increased in skin of S. aureus-infected patients. We next developed a S. aureus cutaneous infection mouse model and found that IL-33 was significantly increased in dermal macrophages of infected mouse skin. The expression of IL-33 by macrophages was induced by staphylococcal peptidoglycan (PGN) and lipoteichoic acid (LTA) via activation of toll-like receptor 2(TLR2) –mitogen-activated protein kinase (MAPK)-AKT-signal transducer and activator of transcription 3(STAT3) signaling pathway as PGN and LTA failed to induce IL-33 in Tlr2-deficient peritoneal macrophages, and MAPK,AKT, STAT3 inhibitors significantly decreased PGN- or LTA-induced IL-33. IL-33, in turn, acted on macrophages to induce microbicidal nitric oxygen (NO) release. This induction was dependent on inducible nitric oxide synthase (iNOS) activation, as treatment of macrophages with an inhibitor of iNOS, aminoguanidine, significantly decreased IL-33-induced NO release. Moreover, aminoguanidine significantly blocked the capacity of IL-33 to inhibit the growth of S. aureus, and IL-33 silencing in macrophages significantly increased the survival of S. aureus in macrophages. Furthermore, the administration of IL-33-neutralizing antibody into mouse skin decreased iNOS production but increased the survival of S. aureus in skin. These findings reveal that IL-33 can promote antimicrobial capacity of dermal macrophages, thus enhancing antimicrobial defense against skin bacterial infections.
Author Summary
Interleukin-33 (IL-33) is associated with multiple diseases, including asthma, rheumatoid arthritis, tissue injuries and infections. Although IL-33 has been indicated to be involved in wound infections, little is known about how IL-33 is regulated as a mechanism to increase host defense against skin bacterial infections. Here we have shown that Staphylococcus aureus (S. aureus) cutaneous infection increases IL-33 expression in dermal macrophages in the skin. The expression of IL-33 by macrophages is induced by staphylococcal peptidoglycan (PGN) and lipoteichoic acid (LTA) via activation of toll-like receptor 2(TLR2) –mitogen-activated protein kinase (MAPK)-AKT-signal transducer and activator of transcription 3(STAT3) signaling pathway. IL-33 in turn acts on macrophages to inhibit the growth of S. aureus by binding to its receptor ST2 followed by activation of the AKT-β-catenin pathway, thus inducing and activating inducible nitric oxygen synthase (iNOS) to release microbiocidal nitric oxygen (NO). These findings reveal that IL-33 can promote antimicrobial capacity of dermal macrophages, thus enhancing antimicrobial defense against skin bacterial infections.
doi:10.1371/journal.ppat.1003918
PMCID: PMC3930573  PMID: 24586149
15.  Treatment with Pyranopyran-1, 8-Dione Attenuates Airway Responses in Cockroach Allergen Sensitized Asthma in Mice 
PLoS ONE  2014;9(1):e87558.
Chronic allergic asthma is characterized by Th2-typed inflammation, and contributes to airway remodeling and the deterioration of lung function. Viticis Fructus (VF) has long been used in China and Korea as a traditional herbal remedy for treating various inflammatory diseases. Previously, we have isolated a novel phytochemical, pyranopyran-1, 8-dione (PPY), from VF. This study was conducted to evaluate the ability of PPY to prevent airway inflammation and to attenuate airway responses in a cockroach allergen-induced asthma model in mice. The mice sensitized to and challenged with cockroach allergen were treated with oral administration of PPY. The infiltration of total cells, eosinophils and lymphocytes into the BAL fluid was significantly inhibited in cockroach allergen-induced asthma mice treated with PPY (1, 2, or 10 mg/kg). Th2 cytokines and chemokine, such as IL-4, IL-5, IL-13 and eotaxin in BAL fluid were also reduced to normal levels following treatment with PPY. In addition, the levels of IgE were also markedly suppressed after PPY treatment. Histopathological examination demonstrated that PPY substantially inhibited eosinophil infiltration into the airway, goblet cell hyperplasia and smooth muscle hypertrophy. Taken together, these results demonstrate that PPY possesses a potent efficacy on controlling allergic asthma response such as airway inflammation and remodeling.
doi:10.1371/journal.pone.0087558
PMCID: PMC3906187  PMID: 24489937
16.  Dual Targeting of MEK and PI3K Pathways Attenuates Established and Progressive Pulmonary Fibrosis 
PLoS ONE  2014;9(1):e86536.
Pulmonary fibrosis is often triggered by an epithelial injury resulting in the formation of fibrotic lesions in the lung, which progress to impair gas exchange and ultimately cause death. Recent clinical trials using drugs that target either inflammation or a specific molecule have failed, suggesting that multiple pathways and cellular processes need to be attenuated for effective reversal of established and progressive fibrosis. Although activation of MAPK and PI3K pathways have been detected in human fibrotic lung samples, the therapeutic benefits of in vivo modulation of the MAPK and PI3K pathways in combination are unknown. Overexpression of TGFα in the lung epithelium of transgenic mice results in the formation of fibrotic lesions similar to those found in human pulmonary fibrosis, and previous work from our group shows that inhibitors of either the MAPK or PI3K pathway can alter the progression of fibrosis. In this study, we sought to determine whether simultaneous inhibition of the MAPK and PI3K signaling pathways is a more effective therapeutic strategy for established and progressive pulmonary fibrosis. Our results showed that inhibiting both pathways had additive effects compared to inhibiting either pathway alone in reducing fibrotic burden, including reducing lung weight, pleural thickness, and total collagen in the lungs of TGFα mice. This study demonstrates that inhibiting MEK and PI3K in combination abolishes proliferative changes associated with fibrosis and myfibroblast accumulation and thus may serve as a therapeutic option in the treatment of human fibrotic lung disease where these pathways play a role.
doi:10.1371/journal.pone.0086536
PMCID: PMC3903543  PMID: 24475138
17.  Peripheral Erythrocytes Decrease upon Specific Respiratory Challenge with Grass Pollen Allergen in Sensitized Mice and in Human Subjects 
PLoS ONE  2014;9(1):e86701.
Background and Aims
Specific hyper-responsiveness towards an allergen and non-specific airway hyperreactivity both impair quality of life in patients with respiratory allergic diseases. We aimed to investigate cellular responses following specific and non-specific airway challenges locally and systemically in i) sensitized BALB/c mice challenged with grass pollen allergen Phl p 5, and in ii) grass pollen sensitized allergic rhinitis subjects undergoing specific airway challenge in the Vienna Challenge Chamber (VCC).
Methods and Results
BALB/c mice (n = 20) were intraperitoneally immunized with grass pollen allergen Phl p 5 and afterwards aerosol challenged with either the specific allergen Phl p 5 (n = 10) or the non-specific antigen ovalbumin (OVA) (n = 10). A protocol for inducing allergic asthma as well as allergic rhinitis, according to the united airway concept, was used. Both groups of exposed mice showed significantly reduced physical activity after airway challenge. Specific airway challenge further resulted in goblet cell hyperplasia, enhanced mucous secretion, intrapulmonary leukocyte infiltration and lymphoid follicle formation, associated with significant expression of IL-4, IL-5 and IL-13 in splenocytes and also partially in lung tissue. Concerning circulating blood cell dynamics, we observed a significant drop of erythrocyte counts, hemoglobin and hematocrit levels in both mouse groups, challenged with allergen or OVA. A significant decrease in circulating erythrocytes and hematocrit levels after airway challenges with grass pollen allergen was also found in grass pollen sensitized human rhinitis subjects (n = 42) at the VCC. The effects on peripheral leukocyte counts in mice and humans however were opposed, possibly due to the different primary inflammation sites.
Conclusion
Our data revealed that, besides significant leukocyte dynamics, particularly erythrocytes are involved in acute hypersensitivity reactions to respiratory allergens. A rapid recruitment of erythrocytes to the lungs to compensate for hypoxia is a possible explanation for these findings.
doi:10.1371/journal.pone.0086701
PMCID: PMC3899302  PMID: 24466205
18.  Galectin-9 Enhances Cytokine Secretion, but Suppresses Survival and Degranulation, in Human Mast Cell Line 
PLoS ONE  2014;9(1):e86106.
Galectin-9 (Gal-9), a lectin having a β-galactoside-binding domain, can induce apoptosis of Th1 cells by binding to TIM-3. In addition, Gal-9 inhibits IgE/Ag-mediated degranulation of mast cell/basophilic cell lines by binding to IgE, thus blocking IgE/Ag complex formation. However, the role of Gal-9 in mast cell function in the absence of IgE is not fully understood. Here, we found that recombinant Gal-9 directly induced phosphorylation of Erk1/2 but not p38 MAPK in a human mast cell line, HMC-1, which does not express FcεRI. Gal-9 induced apoptosis and inhibited PMA/ionomycin-mediated degranulation of HMC-1 cells. On the other hand, Gal-9 induced cytokine and/or chemokine production by HMC-1 cells, dependent on activation of ERK1/2 but not p38 MAPK. In addition, the lectin activity of Gal-9 was required for Gal-9-mediated cytokine secretion by HMC-1 cells. These observations suggest that Gal-9 has dual properties as both a regulator and an activator of mast cells.
doi:10.1371/journal.pone.0086106
PMCID: PMC3896437  PMID: 24465902
19.  Preventive Effect of Halofuginone on Concanavalin A-Induced Liver Fibrosis 
PLoS ONE  2013;8(12):e82232.
Halofuginone (HF) is an active component of extracts derived from the plant alkaloid febrifugine and has shown therapeutic promise in animal models of fibrotic disease. Our main objectives were to clarify the suppressive effect of HF on concanavalin A (ConA)-induced liver fibrosis. ConA injection into the tail vein caused a great increase in the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, while orally administration of HF significantly decreased the levels of the transaminases. In addition, the levels of hyaluronic acid (HA), procollagen III (PCIII) and TGF-β1 in the serum and collagen I, α-SMA, tissue inhibitors of metalloproteinase 2 (TIMP2) and Smad3 in the liver tissue were significantly lowered with the treatment of HF. Histological examination also demonstrated that HF significantly reduced the severity of liver fibrosis. Since ConA-induced liver fibrosis is caused by the repeated activation of T cells, immunomodulatory substances might be responsible for the suppressive effect of HF. We found that the production of nuclear factor (NF)-kB in the serum was increased in ConA-treated group, while decreased significantly with the treatment of HF. The changes of inflammatory cytokines tumor necrosis factor (TNF-α), IL-6 and IL-1β in the serum followed the same rhythm. All together, our findings indicate that orally administration HF (10ppm) would attenuate the liver fibrosis by suppressing the synthesis of collagen I and inflammation-mediated liver injury.
doi:10.1371/journal.pone.0082232
PMCID: PMC3864948  PMID: 24358159
20.  Alveolar Macrophage Innate Response to Mycobacterium immunogenum, the Etiological Agent of Hypersensitivity Pneumonitis: Role of JNK and p38 MAPK Pathways 
PLoS ONE  2013;8(12):e83172.
Mycobacterium immunogenum is an emerging pathogen of the immune-mediated lung disease hypersensitivity pneumonitis (HP) reported in machinists occupationally exposed to contaminated metal working fluid (MWF). However, the mechanism of its interaction with the host lung is unclear. Considering that alveolar macrophages play a central role in host defense in the exposed lung, understanding their interaction with the pathogen could provide initial insights into the underlying immunopathogenesis events and mechanisms. In the current study, M. immunogenum 700506, a predominant genotype isolated from HP-linked fluids, was shown to multiply intracellularly, induce proinflammatory mediators (TNF-α, IL-1α, IL-1β, IL-6, GM-CSF, NO) and cause cytotoxicity/cell death in the cultured murine alveolar macrophage cell line MH-S in a dose- and time-dependent manner. The responses were detected as early as 3h post-infection. Comparison of this and four additional genotypes of M. immunogenum (MJY-3, MJY-4, MJY-12, MJY-14) using an effective dose-time combination (100 MOI for 24h) showed these macrophage responses in the following order (albeit with some variations for individual response indicators). Inflammatory: MJY-3 ≥ 700506 > MJY-4 ≥ MJY-14 ≥ MJY-12; Cytotoxic: 700506 ≥ MJY-3 > MJY-4 ≥ MJY-12 ≥ MJY-14. In general, 700506 and MJY-3 showed a more aggressive response than other genotypes. Chemical blocking of either p38 or JNK inhibited the induction of proinflammatory mediators (cytokines, NO) by 700506. However, the cellular responses showed a somewhat opposite effect. This is the first report on M. immunogenum interactions with alveolar macrophages and on the identification of JNK- and p38- mediated signaling and its role in mediating the proinflammatory responses during these interactions.
doi:10.1371/journal.pone.0083172
PMCID: PMC3859638  PMID: 24349452
21.  MHC Class II Expression in Human Basophils: Induction and Lack of Functional Significance 
PLoS ONE  2013;8(12):e81777.
The antigen-presenting abilities of basophils and their role in initiating a Th2 phenotype is a topic of current controversy. We aimed to determine whether human basophils can be induced to express MHC Class II and act as antigen presenting cells for T cell stimulation. Isolated human basophils were exposed to a panel of cytokines and TLR-ligands and assessed for MHC Class II expression. MHC Class II was expressed in up to 17% of isolated basophils following incubation with a combination of IL-3, IFN-γ and GM-CSF for 72 hours. Costimulatory molecules (CD80 and CD86) were expressed at very low levels after stimulation. Gene expression analysis of MHC Class II-positive basophils confirmed up-regulation of HLA-DR, HLA-DM, CD74 and Cathepsin S. However, MHC Class II expressing basophils were incapable of inducing antigen-specific T cell activation or proliferation. This is the first report of significant cytokine-induced MHC Class II up-regulation, at both RNA and protein level, in isolated human basophils. By testing stimulation with relevant T cell epitope peptide as well as whole antigen, the failure of MHC Class II expressing basophils to induce T cell response was shown not to be solely due to inefficient antigen uptake and/or processing.
doi:10.1371/journal.pone.0081777
PMCID: PMC3857240  PMID: 24349126
22.  The Transcriptomic Response of Rat Hepatic Stellate Cells to Endotoxin: Implications for Hepatic Inflammation and Immune Regulation 
PLoS ONE  2013;8(12):e82159.
With their location in the perisinusoidal space of Disse, hepatic stellate cells (HSCs) communicate with all of the liver cell types both by physical association (cell body as well as cytosolic processes penetrating into sinusoids through the endothelial fenestrations) and by producing several cytokines and chemokines. Bacterial lipopolysaccharide (LPS), circulating levels of which are elevated in liver diseases and transplantation, stimulates HSCs to produce increased amounts of cytokines and chemokines. Although recent research provides strong evidence for the role of HSCs in hepatic inflammation and immune regulation, the number of HSC-elaborated inflammatory and immune regulatory molecules may be much greater then known at the present time. Here we report time-dependent changes in the gene expression profile of inflammatory and immune-regulatory molecules in LPS-stimulated rat HSCs, and their validation by biochemical analyses. LPS strongly up-regulated LPS-response elements (TLR2 and TLR7) but did not affect TLR4 and down-regulated TLR9. LPS also up-regulated genes in the MAPK, NFκB, STAT, SOCS, IRAK and interferon signaling pathways, numerous CC and CXC chemokines and IL17F. Interestingly, LPS modulated genes related to TGFβ and HSC activation in a manner that would limit their activation and fibrogenic activity. The data indicate that LPS-stimulated HSCs become a major cell type in regulating hepatic inflammatory and immunological responses by altering expression of numerous relevant genes, and thus play a prominent role in hepatic pathophysiology including liver diseases and transplantation.
doi:10.1371/journal.pone.0082159
PMCID: PMC3857241  PMID: 24349206
23.  Spironolactone Attenuates Bleomycin-Induced Pulmonary Injury Partially via Modulating Mononuclear Phagocyte Phenotype Switching in Circulating and Alveolar Compartments 
PLoS ONE  2013;8(11):e81090.
Background
Recent experimental studies provide evidence indicating that manipulation of the mononuclear phagocyte phenotype could be a feasible approach to alter the severity and persistence of pulmonary injury and fibrosis. Mineralocorticoid receptor (MR) has been reported as a target to regulate macrophage polarization. The present work was designed to investigate the therapeutic potential of MR antagonism in bleomycin-induced acute lung injury and fibrosis.
Methodology/Principal Findings
We first demonstrated the expression of MR in magnetic bead-purified Ly6G-/CD11b+ circulating monocytes and in alveolar macrophages harvested in bronchoalveolar lavage fluid (BALF) from C57BL/6 mice. Then, a pharmacological intervention study using spironolactone (20mg/kg/day by oral gavage) revealed that MR antagonism led to decreased inflammatory cell infiltration, cytokine production (downregulated monocyte chemoattractant protein-1, transforming growth factor β1, and interleukin-1β at mRNA and protein levels) and collagen deposition (decreased lung total hydroxyproline content and collagen positive area by Masson’ trichrome staining) in bleomycin treated (2.5mg/kg, via oropharyngeal instillation) male C57BL/6 mice. Moreover, serial flow cytometry analysis in blood, BALF and enzymatically digested lung tissue, revealed that spironolactone could partially inhibit bleomycin-induced circulating Ly6Chi monocyte expansion, and reduce alternative activation (F4/80+CD11c+CD206+) of mononuclear phagocyte in alveoli, whereas the phenotype of interstitial macrophage (F4/80+CD11c-) remained unaffected by spironolactone during investigation.
Conclusions/Significance
The present work provides the experimental evidence that spironolactone could attenuate bleomycin-induced acute pulmonary injury and fibrosis, partially via inhibition of MR-mediated circulating monocyte and alveolar macrophage phenotype switching.
doi:10.1371/journal.pone.0081090
PMCID: PMC3834272  PMID: 24260540
24.  Proton-Sensing Ovarian Cancer G Protein-Coupled Receptor 1 on Dendritic Cells Is Required for Airway Responses in a Murine Asthma Model 
PLoS ONE  2013;8(11):e79985.
Ovarian cancer G protein-coupled receptor 1 (OGR1) stimulation by extracellular protons causes the activation of G proteins and subsequent cellular functions. However, the physiological and pathophysiological roles of OGR1 in airway responses remain largely unknown. In the present study, we show that OGR1-deficient mice are resistant to the cardinal features of asthma, including airway eosinophilia, airway hyperresponsiveness (AHR), and goblet cell metaplasia, in association with a remarkable inhibition of Th2 cytokine and IgE production, in an ovalbumin (OVA)-induced asthma model. Intratracheal transfer to wild-type mice of OVA-primed bone marrow-derived dendritic cells (DCs) from OGR1-deficient mice developed lower AHR and eosinophilia after OVA inhalation compared with the transfer of those from wild-type mice. Migration of OVA-pulsed DCs to peribronchial lymph nodes was also inhibited by OGR1 deficiency in the adoption experiments. The presence of functional OGR1 in DCs was confirmed by the expression of OGR1 mRNA and the OGR1-sensitive Ca2+ response. OVA-induced expression of CCR7, a mature DC chemokine receptor, and migration response to CCR7 ligands in an in vitro Transwell assay were attenuated by OGR1 deficiency. We conclude that OGR1 on DCs is critical for migration to draining lymph nodes, which, in turn, stimulates Th2 phenotype change and subsequent induction of airway inflammation and AHR.
doi:10.1371/journal.pone.0079985
PMCID: PMC3823589  PMID: 24244587
25.  Chlamydia pneumoniae Infection in Mice Induces Chronic Lung Inflammation, iBALT Formation, and Fibrosis 
PLoS ONE  2013;8(10):e77447.
Chlamydia pneumoniae (CP) lung infection can induce chronic lung inflammation and is associated with not only acute asthma but also COPD exacerbations. However, in mouse models of CP infection, most studies have investigated specifically the acute phase of the infection and not the longer-term chronic changes in the lungs. We infected C57BL/6 mice with 5×105 CP intratracheally and monitored inflammation, cellular infiltrates and cytokine levels over time to investigate the chronic inflammatory lung changes. While bacteria numbers declined by day 28, macrophage numbers remained high through day 35. Immune cell clusters were detected as early as day 14 and persisted through day 35, and stained positive for B, T, and follicular dendritic cells, indicating these clusters were inducible bronchus associated lymphoid tissues (iBALTs). Classically activated inflammatory M1 macrophages were the predominant subtype early on while alternatively activated M2 macrophages increased later during infection. Adoptive transfer of M1 but not M2 macrophages intratracheally 1 week after infection resulted in greater lung inflammation, severe fibrosis, and increased numbers of iBALTS 35 days after infection. In summary, we show that CP lung infection in mice induces chronic inflammatory changes including iBALT formations as well as fibrosis. These observations suggest that the M1 macrophages, which are part of the normal response to clear acute C. pneumoniae lung infection, result in an enhanced acute response when present in excess numbers, with greater inflammation, tissue injury, and severe fibrosis.
doi:10.1371/journal.pone.0077447
PMCID: PMC3808399  PMID: 24204830

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