Experimental autoimmune encephalomyelitis (EAE) is an animal model that captures many of the hallmarks of human multiple sclerosis (MS), including blood–brain barrier (BBB) breakdown, inflammation, demyelination and axonal destruction. The standard clinical score measurement of disease severity and progression assesses functional changes in animal mobility; however, it does not offer information regarding the underlying pathophysiology of the disease in real time. The purpose of this study was to apply a novel optical imaging technique that offers the advantage of rapid imaging of relevant biomarkers in live animals.139-151-induced EAE and in response to tolerogenic therapy.Advances in non-invasive fluorescence molecular tomographic (FMT) imaging, in combination with a variety of biological imaging agents, offer a unique, sensitive and quantifiable approach to assessing disease biology in living animals. Using vascular (AngioSense 750EX) and protease-activatable cathepsin B (Cat B 680 FAST) near infrared (NIR) fluorescence imaging agents to detect BBB breakdown and inflammation, respectively, we quantified brain and spinal cord changes in mice with relapsing-remitting PLPex vivo tissue imaging and comparison to clinical score results and histopathological analysis of CNS tissue. FMT imaging showed clear differences between control and diseased mice, and immune tolerance induction by antigen-coupled PLGA nanoparticles effectively blocked both disease induction and accumulation of imaging agents in the brain and spinal cord.FMT imaging and analysis techniques were carefully characterized and non-invasive imaging results corroborated by both Cat B 680 FAST and AngioSense 750EX offered the combination best able to detect disease in both the brain and spinal cord, as well as the downregulation of disease by antigen-specific tolerance. Non-invasive optical tomographic imaging thus offers a unique approach to monitoring neuroinflammatory disease and therapeutic intervention in living mice with EAE. The experimental autoimmune encephalomyelitis (EAE) model of MS captures many of the hallmarks of the human disease ,2. EAE cEAE is generally assessed using a subjective clinical score assessment, which does not provide information about underlying cellular or molecular processes and does not always reflect the underlying pathological changes . AdditioTM, ProSense® 750EX and MMPSense® 750 FAST), enzymatic renin activity associated with abnormal renin-angiotensin-system activity in the CNS (ReninSense 680 FASTTM) and vascular agents designed to detect blood–brain barrier breakdown . Imaging and analysis techniques were carefully characterized and non-invasive imaging results corroborated by both ex vivo tissue imaging and comparison to clinical score results. Cat B 680 FAST and AngioSense 750EX offered a combination best able to detect disease in the brain and spinal cord, as well as the downregulation of disease by antigen-specific tolerance.Optical imaging is a relatively new modality and offers the potential advantage of rapid imaging of biological markers. Approaches with bioluminescence and fluorescence in demyelination research have relied on either transgenic luciferase-expressing mice  or syste139-151-induced experimental autoimmune encephalomyelitis experiments, specific pathogen-free female SJL/J mice (6 to 8 weeks of age) were purchased from Harlan Laboratories  and housed at the Center for Comparative Medicine at Northwestern University  under a controlled environment  under specific pathogen-free conditions with water and food provided ad libitum. All experiments were performed in accordance with Northwestern University IACUC guidelines for animal care and use. Proteolipid protein (PLP)139–151 (HSLGKWLGHPDKF) and ovalbumin (OVA)323–339 (ISQAVHAAHAEINEAGR) were purchased from Genemed Synthesis. EAE was elicited by immunization with 50 μg of PLP139-151 in complete Freund’s adjuvant (CFA) supplemented with 200 μg heat-killed M. tuberculosis H37Ra . A volume of 0.1 ml of emulsion was distributed subcutaneously over three spots on the dorsal flanks on day 0. Observational clinical scores for each mouse were recorded daily using a scale of 0–5 as listed below:For the PLPClinical Score of 0: No abnormalitiesClinical Score of 1: Limp tail or hind limb weaknessClinical Score of 2: Both limp tail and hind limb weaknessClinical Score of 3: Partial hind limb paralysisClinical Score of 4: Total hind limb paralysisClinical Score of 5: Moribund9 nanoparticles comprising 10–15 μg of peptide, depending on the peptide sequence used in the coupling reaction.As previously described , 500-nm Six commercially available imaging agents  were used to optimize EAE imaging and detect therapeutic efficacy Table . AngioSein vivo imaging system . For whole body imaging, the anesthetized mouse was placed in a prone position on the imaging cassette to capture the spine or brain of the animal within the imaging scan field. The imaging cassette was adjusted to the proper depth to gently restrain each mouse and then inserted into the heated docking system (regulated at ~37°C) in the FMT imaging chamber. For imaging the head, the mouse was positioned prone on a resin block  and positioned in the imaging cassette. A NIR laser diode was used to transilluminate  each mouse’s body, with signal detection occurring via a thermoelectrically cooled CCD camera placed on the opposite side of the imaged animal. Appropriate optical filters allowed collection of both fluorescence and excitation data sets, and the multiple source-detector fluorescence projections were normalized to the paired collection of laser excitation data. The entire image acquisition sequence took approximately 6–8 min per mouse. FMT 4000 epifluorescence imaging was routinely performed prior to each tomographic imaging session using built-in LED front illuminators and collection of single camera images.Mice were maintained on a low fluorescence alfalfa-free diet Harlan 2019) recommended for fluorescence imaging, and imaging was performed at the peak of disease (d15). The mice were injected intravenously with 2× the recommended dose of fluorogenic agents (to facilitate brain biodistribution) at d14 and imaged 24 h later  for the quantification of fluorescence signal within the head and spine. Three-dimensional regions of interest (ROI) were drawn to encompass each region, and a modest threshold was applied identically to all animals within each agent group in order to minimize background signal. Briefly, the brain ROI for each mouse was placed as a 9 × 12 × 6-mm (w × h × d) ellipsoid in the appropriate head region, and a threshold was applied equal to 30% of the average maximum fluorescence (in nM) of the control brain ROIs. For spinal cord ROIs, each was placed as a 6 × 31 × 4-mm rectangular prism with thresholding applied as 10% of the average maximum fluorescence (nM) of the control spinal cord ROIs. The total amount of brain and spinal cord fluorescence (in pmoles) was automatically calculated relative to internal standards generated with known concentrations of the appropriate NIR dyes. For visualization and analysis purposes, TrueQuant software provided 3D images and tomographic slices.The collected fluorescence data sets were reconstructed by FMT 4000 system software  as described in the Materials and Methods section. Injection sites (3 per mouse) were placed laterally on the backs of the mice to avoid interference with spinal cord imaging. PLP peptide-immunized mice achieved mild disease  with 60% disease incidence. Mice were injected with 4 nmol of AS750 when disease severity in half of the mice achieved a clinical score of 2–3 (day 14), and imaging was performed 24 h later to allow optimal accumulation of the agent in BBB-compromised areas of the CNS.To assess whether mouse EAE can be effectively imaged by FMT, we used an NIR vascular imaging agent, AngioSense 750EX (AS750), to detect vascular leaks in the blood–brain barrier associated with CNS disease. On day zero, SJL/J mice were immunized with PLP139-151-immunized mouse, revealing vascular leak within the resulting granulomas. Transillumination scan fields (required for FMT tomographic imaging) were positioned based upon the epifluorescence images in order to capture the head and spinal cord in two separate 2–4-min scans , and 60% showed elevated spine signal. These non-invasive imaging results were confirmed by ex vivo epifluorescence imaging and quantification of excised brain and spinal cord tissue  and brain 3 mm depth). The resulting slice images . Mice were imaged and analyzed as described in the study shown in Figures We next examined the potential of assessing protease activity as a biomarker of EAE severity by imaging mice at peak of acute disease with protease-activatable NIR fluorescence imaging agents: Cat B 680 FAST (CB680) for cathepsin B activity, ProSense 750EX (PS750) for pan-cathepsin activity , MMPSense 750 FAST (MMP750) for pan-matrix metalloprotease activity , and ReninSense 680 FAST (RS680) for renin activity. The average clinical score of all PLPex vivo tissue imaging. Interestingly, neither RS680 nor MMP750 showed significant brain elevations, despite the association of MMPs and renin in EAE and the similar molecular weights as compared to CB680. In contrast, RS680 showed statistically significant elevations in the spinal cord, confirmed by ex vivo tissue assessment. CB680 also showed spinal cord elevation as confirmed both by ex vivo imaging and histological examination showing activated cathepsin B expression in F4/80+ macrophages/microglia in a spinal cord inflammatory lesion , and this was confirmed in 139-151-coupled polystyrene or biodegradable poly(lactic-co-glycolic acid) beads (PLP-PSB or PLP-PLGA) can decrease EAE induction and severity induced by PLP139-151 immunization when using either prophylactic or therapeutic treatment regimens [323-339-PLGA (OVA tolerized) or PLP139-151-PLGA (PLP tolerized) 7 days prior to immunizing mice with PLP139-151/CFA. The average clinical score of control OVA-tolerized mice was 2.4 by day 13, with disease incidence over 70% . Imaging results focused only on the signal within the spinal cord and brain regions for control OVA-tolerized . Brain data, in contrast, showed the inherent variability of brain disease and a lack of correlation with clinical score. Thus, although there was a trend toward a decrease in brain signal with PLP-PLGA treatment, a much larger study would be required to assess this. The ex vivo brain and spinal cord tissue imaging by epifluorescence (right panels) confirmed in vivo observations and showed marginal statistical significance for AS750 brain signal.Quantification of the two-channel fluorescent signal in EAE mice shows excellent detection of therapeutic efficacy of PLP tolerance induction when examining the spinal cord region Figure  of the iRodent models of EAE are used in research and drug development because of their similar pathology and/or pathogenesis to human MS and their ease of use and reproducibility. These models share a number of important morphologic and immunologic features with human MS, and they have contributed greatly to the overall knowledge of immune-mediated myelin destruction. Damage occurs in three phases; first some abnormal areas of tissue damage appear in the brain and spinal cord, followed later by leaks in the blood–brain barrier and then immune cell infiltration ultimately leading to demyelination . EAE disin vivo biological data regarding disease severity, progression and response to therapy. BBB compromise in EAE is well characterized [in vivo imaging of vascular leak into the CNS. In addition, as proteases play a central role in cancer and inflammatory disease processes, we anticipated that protease-activatable NIR imaging agents [New vascular and protease-activatable molecular imaging agents, as well as current improvements in imaging methods, provide an opportunity to gather non-invasive cterized , and Angcterized ,31, offeg agents ,33 couldg agents ,35, asthg agents ,37, atheg agents ,39 and ag agents ,41. HoweFor the first time, we show the benefit of fluorescence molecular tomographic imaging in a mouse model of MS and demonstrate that tomography can not only provide images reflecting the disease severity, but also provide multiplex quantitative measurements Figures , 5 and 6in vivo. Future studies will assess these agents, and additional NIR imaging agents, for their utility to detect disease as well as their sensitivity in detecting very early, subclinical disease.Optical tomographic imaging using NIR imaging agents in EAE has the potential for use in drug discovery research by virtue of deep tissue penetration, quantitative readout (pmol rather than light intensity) and the pairing with NIR imaging agents that detect the cellular participants in the underlying disease pathology. For the past 25 years, the Miller laboratory has explored specific T cell tolerance, induced by antigen-coupled splenocytes, as a means to both treat disease -45 and pin vivo. This technology offers a powerful tool for both basic neuroinflammation research and drug discovery and development by enabling the non-invasive monitoring of cellular processes that also drive human MS pathology. FMT thus should provide a rapid means for assessing and characterizing therapeutics for clinical evaluation.Our studies have built upon advances in imaging technology to establish non-invasive optical tomographic imaging as a robust and accurate means of assessing BBB compromise and neuroinflammation in EAE co-glycolic) acid; PLP: Proteolipid protein; ROI: Regions of interest; SPECT: Single photon emission computed tomography.BBB: Blood–brain barrier; CAT: Cathepsin; CFA: Complete Freund’s adjuvant; EAE: Experimental autoimmune encephalomyelitis; ECDI: 1-Ethyl-3-(3′-dimethylaminopropyl)carbodiimide; FMT: Fluorescence molecular tomographic imaging; MMP: Matrix metalloprotease; MRI: Magnetic resonance imaging; MS: Multiple sclerosis; NIR: Near infrared; OVA: Ovalbumin; PET: Positron emission tomography; PLGA: Poly have been around for several years (and have been published to some extent), the company expects no major financial gain as a result of publication beyond a modest increase in awareness in proportion to the robustness of the research. Northwestern University, Feinberg School of Medicine, employees have no financial stake in PerkinElmer Inc. The authors hold no patents relevant to the manuscript, and no patent applications are being submitted (or planned to be submitted). No reimbursement or funding relating to potential patents has been received. PerkinElmer currently holds patents for the technology used in the research.JDP and SDM designed the research, analyzed data and wrote the paper; VLE, KOV, GEG and ZNH performed the experiments. All authors read and approved the final manuscript.


The observation that pathogenic bacteria are commonly tolerated in the human nose, yet drive florid inflammation in the lung, is poorly understood, partly due to limited availability of primary human cells from each location. We compared responses to bacterial virulence factors in primary human nasal and alveolar cells, and characterised the distribution of Toll-interacting protein  signalling) in the human respiratory tract.Primary cells were isolated from nasal brushings and lung tissue taken from patients undergoing pulmonary resection. Cells were exposed to lipopolysaccharide, lipoteichoic acid, peptidoglycan, CpG-C DNA or tumour necrosis factor (TNF). Cytokines were measured in cell supernatants. TOLLIP was characterised using quantitative real-time PCR and immunofluorescence.In primary alveolar, but not primary nasal, cells peptidoglycan significantly increased secretion of interleukin (IL)-1β, IL-6, IL-8, IL-10 and TNF. TLR2 expression was significantly higher in alveolar cells and correlated with IL-8 production. TOLLIP expression was significantly greater in nasal cells.In conclusion, primary human alveolar epithelial cells are significantly more responsive to peptidoglycan than primary nasal epithelial cells. This may partly be explained by differential TLR2 expression. TOLLIP is expressed widely in the human respiratory tract, and may contribute to the regulation of inflammatory responses. Peptidoglycan exerts a significant proinflammatory cytokine response in primary human alveolar epithelium but not in primary human nasal epithelium.The Toll-like receptor regulator Toll-interacting protein is widely expressed in the human respiratory tract.Hospital-acquired infections (HAIs) are common and associated with significant morbidity and mortality.2Why the nasal epithelium should tolerate these microorganisms well, while the alveolar epithelium mounts such a florid inflammatory response, remains poorly understood. A better understanding of this paradox has been hampered by difficulties in accessing primary cells from the human nose and alveoli.Staphylococcus aureus and Pseudomonas aeruginosa We therefore sought to characterise the effects of key virulence factors from The primary hypothesis for this study was that primary alveolar cells would mount a brisk response to inflammatory stimuli, associated with minimal or absent TOLLIP expression, whereas primary nasal cells would exhibit a blunted response to inflammatory stimuli, associated with abundant TOLLIP expression.Primary human nasal epithelial cells, bronchial epithelial cells and type II alveolar epithelial cells were obtained from patients undergoing elective pneumonectomy or lobectomy for cancer. Methods for obtaining and culturing the nasal and alveolar cells have been described elsewhere.The human colonic carcinoma cell line T84 and the human nasal carcinoma cell line RPMI 2650 were from LGC Promochem . A549 cells  were available in-house.P. aeruginosa strain PA01 , 10 μg/mL of S. aureus peptidoglycan , 10 μg/mL of S. aureus lipoteichoic acid , 10 ng/mL of recombinant human tumour necrosis factor , 1 μΜ CpG-C DNA  or medium alone . Cells were incubated for 24 h at 37°C and supernatants were removed and stored at −80°C until estimation of interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12p70 and TNF assayed using the BD Cytometric Bead Array (CBA) Human Inflammatory Cytokine kit (BD Biosciences), with analysis performed using a BD FACSArray Bioanalyzer System.Confluent cells were treated with 100 ng/mL of ultrapure lipopolysaccharide (LPS) derived from Total RNA was extracted using the total RNA isolation kit Nucleospin RNAII . 1 μg RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit . Primers and probes are summarised in a table in the online supplementary section.Cyclophilin A (PPIA) had the lowest variability rate in the samples assayed. Results were normalised using a TaqMan endogenous control (Applied Biosystems).A Taqman Low Density Array  was used to assess the stability of potential housekeeping genes. Based on the normalisation score, PPIA gene using the standard curve method. Standard curves for the genes of interest were prepared using the plasmids pcDNA3-TLR9-YFP, Addgene plasmid 13642, pcDNA3-TLR4-YFP, Addgene plasmid 13018 and pUC19/human IL-8 Addgene plasmid 17610. Pooled DNA was used in the standard curves for PPIA, TOLLIP and TLR2.Diluted cDNA (1:100) was used as a template for the PCR reaction and samples were loaded onto the Applied Biosystems 7900HT Fast Real-Time PCR System. The specificity of the reactions was controlled using ‘no template’ and ‘no reverse transcription’ controls. Results were normalised to the human Confluent cells were detached using trypsin/EDTA solution (10 min at 37°C), and centrifuged. Resuspended cells were seeded onto glass coverslips for 15 min and incubated overnight at 37°C. Medium was replaced with ice-cold methanol for 10 min, the cells were washed and then blocking was performed using 2% goat serum for 30 min. Cells were dried and antibodies were applied overnight as appropriate: murine monoclonal IgG1 against human cytokeratin 18, murine monoclonal IgG2a against human cytokeratin 19, murine monoclonal IgG2a against human TLR2 , polyclonal rabbit antihuman TLR4 IgG and polyclonal rabbit antihuman TOLLIP IgG (Abcam). Controls comprised murine isotype monoclonal antibodies (Invitrogen) or, where polyclonal primaries were used, non-immune rabbit IgG (Invitrogen). The following day cells were washed with phosphate buffered saline and secondary antibodies applied for 1 h. Secondary antibodies comprised AlexaFluor488-conjugated goat antimouse IgG (Invitrogen) or goat antirabbit IgG conjugated to AlexaFluor 488 (Invitrogen) as appropriate. Cells were washed, dried and Vectashield with DAPI  added. Cells were visualised using a Leica TCS SP5 confocal microscope , and photomicrographs taken.6 cells per well. On the same day 5 mL of Modified Eagles Medium  was inoculated with S. aureus strain Newman, and incubated overnight at 37°C with continuous shaking. The following day an aliquot was inoculated in 5 mL of MEM and allowed to reach logarithmic phase. Bacteria were washed and resuspended in MEM to achieve an optical density of approximately 0.1. Known volumes were (A) added directly to cells and (B) plated onto tryptic soy agar, so that viable bacterial concentrations could be determined by quantifying colony forming units (CFU) the next day. After infection, cells were incubated for a further 4 h at 37°C prior to cell lysis and RNA extraction as above.The cell lines RPMI 2650 or A549 were seeded at a density of 1×10Friedman's test was used to provide a global indication of whether any significant difference existed across the conditions applied to cultured cells. Post hoc analysis comparing unstimulated and stimulated cells was performed using Dunn's test. Comparisons of numerical data between groups were carried out using the Mann-Whitney U test. Comparison of proportions between groups was carried out using Fisher's exact test. Correlations were analysed using Spearman's test. All statistical analyses were performed using GraphPad Prism software . Statistical significance was considered to be at the p<0.05 level.Primary nasal cells were successfully cultured from 6 patients, and primary alveolar cells from 7 . The two groups of patients were similar in their baseline characteristics, although there were more women in the group providing alveolar cells  63% vs 75% of predicted; no significant difference for any of the comparisons). The patients were admitted for resection of non-small cell lung cancer, with the exception of two patients admitted for resection of solitary metastases. Characterisation by quantitative reverse transcriptase PCR (qRT-PCR) demonstrated that cultured nasal epithelial cells consistently expressed the epithelial cell markers cytokeratin 18 and 19 and alveolar epithelial cells expressed the type II pneumocyte markers SP-C and AQP-3 .A range of bacterial virulence factors was applied to primary cells and the cytokine responses were examined by CBA and qRT-PCR. All of the cytokines examined could be produced by primary nasal epithelial cells. However, none of the measured cytokines were significantly upregulated by exposure to PGN, LTA, LPS or CpG . In contAlveolar cell responses were assessed in parallel with nasal cells. LPS and LTA failed to significantly alter secretion of any of the cytokines . HoweverGiven the differential secretion of IL-8 in response to PGN, the effect of this bacterial TLR agonist on IL-8 mRNA production was also analysed. No significant increase in IL-8 expression was observed in either cell type (data not shown), suggesting that at least some of the effect of PGN on IL-8 secretion in alveolar cells may be post-transcriptional.Given that PGN mediates its effects largely through TLR2-mediated recognition and signalling, expression of TLR2 in primary nasal and alveolar epithelial cells was also assessed by qRT-PCR A. TLR2 eTOLLIP expression has been clearly defined in the T84 colonic carcinoma cell lineTOLLIP qRT-PCR assay in this setting. A band of the expected size was consistently detected, and was absent in negative controls  treated under identical conditions. Basal TOLLIP mRNA expression was observed in nasal and alveolar cells but was found to be significantly higher (p<0.05) in the primary nasal epithelial cells  of peripheral accentuation of staining around the cell membrane A–D. PuncTo our knowledge, this study is among the first to compare the differential response of primary human nasal and alveolar epithelial cells to a range of identical inflammatory stimuli, and the first to systematically describe TOLLIP expression and localisation in the human respiratory tract.The findings suggest that primary nasal epithelial cells have a relatively limited repertoire of responsiveness to inflammatory stimuli, generating a statistically significant  increase in the proinflammatory cytokines IL-6 and IL-8, only in response to stimulation with TNF, but not TLR agonists. This responsiveness to TNF is consistent with findings elsewhere.11S. aureus PGN induces a particularly florid inflammatory response in alveolar epithelial cells. It may be particularly relevant that, in our hands, the levels of expression of TLR2 (which recognises PGN) correlated closely with responsiveness, as assessed by IL-8 secretion. The implication seems to be not only that alveolar epithelium expresses more ‘target’ for PGN, but that PGN can upregulate TLR2 expression more effectively on alveolar epithelium. This may go some way to explaining the differential responsiveness of nasal and alveolar epithelium, and perhaps why the lung mounts such a striking inflammatory response to S. aureus, a common ‘coloniser’ of the human nose.In contrast to the relative quiescence of primary nasal cells, we found that primary alveolar epithelial cells were characterised by a more florid response to PGN and TNF that spanned a wider range of cytokines. These observations appear consistent with the hypothesis that bacterial virulence factors are better tolerated by the nose. Our data suggest that TOLLIP mRNA and TOLLIP protein have been detected in commercially available human small airway epithelial cells.TOLLIP mRNA has also been described in pleural effusions.TOLLIP mRNA expression in primary nasal epithelial cells in comparison to type II alveolar epithelial cells broadly supports the hypothesis. The observation that TOLLIP is constitutively and ubiquitously expressed in human respiratory epithelium is consistent with a potential role as a key regulator of inflammatory responses.TOLLIP responsiveness to bacterial virulence factors in nasal and alveolar cell lines.To investigate further potential reasons for differential innate immune responsiveness between the nose and lung, we drew on data describing an excess of TOLLIP in the large intestine, where bacterial tolerance is essential. We believe this to be the first systematic characterisation of TOLLIP's presence and location in primary cells from the human respiratory tract. TOLLIP has been cloned from a human lung cDNA library,TOLLIP binds to IL-1 receptor-associated kinase (IRAK-1), preventing proinflammatory signalling. On stimulation of cells with LPS or IL-1, a receptor complex rapidly forms, incorporating TOLLIP bound to IRAK-1. Sufficient phosphorylation of IRAK-1 allows its dissociation from TOLLIP, and proinflammatory signalling  rapidly ensues. TOLLIP is therefore well placed to regulate inflammatory processes. TOLLIP's ready availability in organs regularly exposed to bacteria, such as the gut, nose and lung, seems potentially important in this regard. Interestingly, TOLLIP has been implicated in LPS hyporesponsiveness in human monocytes and human primary intestinal epithelial cells.21TOLLIP gene that result in reduced TOLLIP function.TOLLIP gene polymorphisms have been weakly associated with increased susceptibility to atopic dermatitis.TOLLIP expression is reduced in tissue from coeliac disease and necrotising enterocolitis.26The functional importance of TOLLIP as a regulator of acute inflammation is supported by emerging clinical data. For example, in the Chinese Han population, increased susceptibility to sepsis is conferred by polymorphisms in the While the data here are some way from having direct clinical relevance, validation of a florid alveolar response to PGN in other cohorts might yield avenues for further exploration. In particular, selective administration of anti-TLR2 or specific TLR regulators early in the florid proinflammatory phase of staphylococcal pneumonia seems theoretically attractive in a condition with continued high mortality despite modern antibiotics and supportive care. The association between TLR2 expression and IL-8 secretion in unstimulated and PGN-stimulated cells is potentially relevant in this regard.Comparison of responses in primary human cells increases the relevance of this study. However, we recognise that there are several potential limitations. First, all of our patients had cancer and most had a long history of smoking, which is known to affect cytokine secretion by epithelial cells throughout the respiratory tract.29In summary, primary human alveolar epithelial cells appear to mount a more exuberant inflammatory response to PGN and TNF than do primary human nasal epithelial cells. PGN's effects may relate to the relative abundance and regulation of TLR2 in the upper and lower airway. TOLLIP is produced throughout the human respiratory tract. TOLLIP is expressed in greater levels in nasal cells than in alveolar epithelial cells, but differential TOLLIP expression in nasal and lung cells in response to bacterial virulence factors was not observed. These data suggest that relative expression of TLR2 and TOLLIP might play a role in the tolerant nature of the nasal epithelium to bacteria. Further studies are required to address a range of remaining questions—these include, but are by no means limited to: whether other TLR regulators are differentially expressed (constitutively or inducibly) in nasal versus alveolar epithelium; whether bacterial virulence factors differentially influence TLR regulator expression within alveolar epithelial cells (favouring a proinflammatory effect of PGN but not the other virulence factors measured here) and whether PGN can evade membrane-based TLR regulators on alveolar cells.


These novel egg loggers revealed high-resolution, three-dimensional egg turning behavior heretofore never measured in wild birds. This new form of biotechnology has broad applicability for addressing fundamental questions in avian breeding ecology, life history, and development, and can be used as a tool to monitor birds that are sensitive to disturbance while breeding.Egg turning is unique to birds and critical for embryonic development in most avian species. Technology that can measure changes in egg orientation and temperature at fine temporal scales (1 Hz) was neither readily available nor small enough to fit into artificial eggs until recently. Here we show the utility of novel miniature data loggers equipped with 3-axis  accelerometers, magnetometers, and a temperature thermistor to study egg turning behavior in free-ranging birds. Artificial eggs containing egg loggers were deployed in the nests of three seabird species for 1–7 days of continuous monitoring. These species (1) turned their eggs more frequently (up to 6.5 turns h The vast majority of bird species exhibit disparate incubation behaviors from their Theropod ancestors, most markedly in their parental physical contact with a clutch of eggs Ptychoramphus aleuticus), western gulls , and Laysan albatrosses (Phoebastria immutabilis). These species represent a wide range in body masses (250–3000 g), egg masses (27–300 g), and clutch sizes (1–3 eggs), see Table S1 in New microtechnologies incorporated into smart phones and tablet computers  have facilitated a variety of studies that examine body orientation and characterize movements of free-ranging animals All animal research was conducted in accordance with approvals from San Jose State University Institutional Animal Care and Use Committee , Pacific Rim Conservation, Point Blue Conservation Science, California Parks, and the UC Natural Reserve System. Bird Banding permits, Migratory Bird Treaty Act permits, and Special Use Permits for all research were granted by the US Fish and Wildlife Service, US Geological Survey, and the State of Hawaii.Pilot tests of loggers housed in artificial eggs were performed prior to conducting field deployments. Temperature measurement accuracy and battery life of the egg loggers were tested and verified using a standard poultry incubator with automatic egg turner , in which egg ambient temperature  and rotation were monitored using visual observations (1–3 times daily) for periods of 24 to 240 hours. Changes in egg logger orientation were tested and verified by comparing a video recording of an egg being turned manually with an animation created by post-processing the data collected by the egg logger.Egg loggers were deployed in the nests of each species for 2–12 days in 2012 . Study nData loggers containing 3-axis accelerometers and magnetometers capable of sensing 1–2° angular changes in roll, pitch, and yaw were housed in replicate eggs  of the sensor. The dynamics associated with these movements were filtered out in post-processing, limiting the recorded orientation measurements to those times when the egg was stationary. Therefore, it was possible to accurately resolve the orientation before and after the egg was rotated, but with decreased accuracy during a rotation event.The 3-axis magnetometer measured the direction of the local magnetic field strength, which is a combination of the Earth's magnetic field and any nearby disturbances. Magnetic disturbances due to the egg itself (hard and soft iron effects) were calibrated during post-processing see . Externa2 resolution), 3-axis magnetometer (0.2 µT resolution), and a temperature thermistor . Given that loggers were centered inside the eggs, temperature readings were considered to be core egg temperature and not peripheral temperature. Logger operation was controlled by a Microchip PIC24F32K302 microcontroller, which triggered measurements once per second. Raw sensor measurement data were recorded to a μSD card at a rate of approximately 115 KB per hour. A 60-mAh single cell Lithium Polymer battery with a low-voltage protection circuit powered the loggers. In the field, loggers operated for up to 8 days continuously.Sensors selected for the loggers were contained in ST Microelectronics LSM303DLHC, a single chip with an integrated 3-axis accelerometer . Raw accelerometer and magnetometer measurements were converted to 3-2-1 Euler angles  produced jagged step changes in egg temperatures over short temporal periods . ConsequBecause egg loggers recorded continuously for several days, we evaluated whether egg turning rates differed between day and night using ephemeris tables to determine the time of local sunrise and sunset at each colony. Colony latitude and longitude , western gulls (N = 17), and Laysan albatrosses  for durations averaging 3.5 to 7.2 days  for all three species. In each case, egg turning rates were 10–30% higher when magnetometer data were incorporated in the analysis, which equates to 10–20 additional turns of the egg per day , although multiple comparison tests revealed that turning rates only differed between auklets and gulls (P = 0.002). The difference in turning rate between auklets and gulls was likely due to variations in 1) clutch size, because auklets incubate a single egg whereas gulls incubate 1–3 eggs that require greater parental attention, and/or 2) nesting habitat, because auklets nest in burrows that protect against most biological and physical perturbations whereas gulls are surface nesters in large colonies that require greater vigilance from colony disturbances . Albatrosses, which are surface nesters like gulls, but incubate a single egg like auklets, had turning rates intermediate to auklets and gulls  and reported ranges of 1.4–3.2 turns h−1−1, but some of this variability was likely due to the manner in which egg turning was measured. We believe the egg loggers used in this study can provide a standardized method to study egg attendance patterns that allows accurate inter- and intraspecific comparisons of egg turning rates.Comparative studies using similar biotechnology  quantified egg turning rates in Adėlie penguins . We were able to examine diurnal incubation behaviors more closely and predicted that egg turning rates would be lower at night for species not generally active in the colony at night. Indeed, our observations confirm that nesting western gulls and Laysan albatrosses were generally quiescent at night whereas Cassin's auklets followed a nocturnal rhythm where partners exchanged nesting duties. In contrast to what we predicted, turning rates were indistinguishable between night and day for gulls and albatrosses , and S4.Grus spp.; 21) but the pattern was less evident in the auklets and gulls we studied  on the nest Figure S1Logger and its installed orientation next to the replica eggs for each species. The logger axes and egg axes are labeled for each egg. Axis label at the intersection indicates the axis extending in the third dimension to complete a right handed coordinate system.(TIF)Click here for additional data file.Figure S2Visual example of the egg orientation described by 3-2-1 Euler angles, as measured by 3-axis accelerometers and magnetometers placed in artificial eggs and deployed in the nests of wild birds. The egg orientation is achieved by first rotating from North by the yaw angle about the Earth's fixed z-axis (down), followed by rotating by the pitch angle about this intermediate frame's y-axis, and finally rotating by the roll angle about the next intermediate frame's x-axis.(TIF)Click here for additional data file.Figure S3Example of egg temperature smoothing. Egg temperature was recorded every second at a resolution of 0.125°C (blue line). For all subsequent analyses, a smoothing function (i.e. moving average with window size of 5000) was applied to reduce the coarseness of the data. Further details are described in (TIF)Click here for additional data file.Figure S4Representative 24-hour time series showing egg attitude and temperature in a) Cassin's auklet and b) western gull. Shown are the roll, pitch, and yaw Euler angles and corresponding egg temperature in the pane above plotted against local date and time starting at 12.00 noon. Of note is the high turning activity for the Cassin's auklet around 20.30 or dusk, when the partners of incubating birds return to the colony each night to exchange incubating duties.(TIF)Click here for additional data file.File S1Supplemental methods. Additional methods, data analysis and processing of egg logger data.(DOCX)Click here for additional data file.File S2Animation of egg turning in a Cassin's auklet.(MP4)Click here for additional data file.File S3Animation of egg turning in a western gull.(MP4)Click here for additional data file.File S4Animation of egg turning in a Laysan albatross.(MP4)Click here for additional data file.


Fusarium graminearum is an opportunistic pathogen of cereals where it causes severe yield losses and concomitant mycotoxin contamination of the grains. The pathogen has mixed biotrophic and necrotrophic (saprophytic) growth phases during infection and the regulatory networks associated with these phases have so far always been analyzed together. In this study we compared the transcriptomes of fungal cells infecting a living, actively defending plant representing the mixed live style (pathogenic growth on living flowering wheat heads) to the response of the fungus infecting identical, but dead plant tissues (cold-killed flowering wheat heads) representing strictly saprophytic conditions. We found that the living plant actively suppressed fungal growth and promoted much higher toxin production in comparison to the identical plant tissue without metabolism suggesting that molecules signaling secondary metabolite induction are not pre-existing or not stable in the plant in sufficient amounts before infection. Differential gene expression analysis was used to define gene sets responding to the active or the passive plant as main impact factor and driver for gene expression. We correlated our results to the published F. graminearum transcriptomes, proteomes, and secretomes and found that only a limited number of in planta- expressed genes require the living plant for induction but the majority uses simply the plant tissue as signal. Many secondary metabolite (SM) gene clusters show a heterogeneous expression pattern within the cluster indicating that different genetic or epigenetic signals govern the expression of individual genes within a physically linked cluster. Our bioinformatic approach also identified fungal genes which were actively repressed by signals derived from the active plant and may thus represent direct targets of the plant defense against the invading pathogen. Fusarium graminearum (telemorph: Giberella zeae) is a plant pathogenic ascomycete fungus causing various plant diseases on small-grain cereals such as Fusarium head blight (FHB or scrab) of wheat (Triticium aestivum) and barley (Hordeum vulgare) as well as ear and stalk rot of maize  and zearalenone , Gβ (GzGPB1), Gγ (GzGPG1), and the Ras-GTPase RAS2 . Some of these conditions can be mimicked in vitro in axenic liquid shake cultures and in fact induction of DON (and it's acetylated derivatives 15ADON and 3ADON) was found in the presence of compounds which are part of the polyamine and urea cycle pathways in plants  and during different stages of infection on wheat and barley  while others may be directly involved in the pathogenic process (overcoming resistance). In order to better define those fungal genes which are directly associated with pathogenicity by counteracting plant defense we compared in this study F. graminearum transcriptomes originating from Ph-1 wild type cultures growing on living flowering wheat heads (pathogenic conditions) or on identical, but cold-killed material (saprophytic conditions). For this comparison we inoculated ears of flowering wheat heads but one half of them had been inactivated prior to infection by cutting them off the plant and dipping them into liquid nitrogen. Both samples were further incubated under the same conditions thus presenting to the fungal cells basically identical substrates but in one case under pathogenic conditions on the living “active” plant and in the other case under saprophytic conditions on the cold-killed “passive” plant material. In this paper we show that only background levels of secondary metabolites are formed on the dead plant material and that this approach is able to differentiate pathogenicity related genes from plant matrix genes and also found genes repressed by the active plant potentially revealing novel fungal targets of active plant defense.It has been recognized only recently from our work and others that virulence factors such as secondary metabolites or effector proteins are not only under genetic, but also under epigenetic control in various fungi, including Remus representing the interaction of the fungus with the living host. This condition is subsequently referred to as “pathogenic growth.” Another set of three ears, which were cut off the plant and shock-frozen in liquid nitrogen prior to spore application, was identically inoculated representing the same plant substrate but without active metabolism and defense responses. We called this condition subsequently “saprophytic growth.” The inoculated living wheat heads were cultivated under standard conditions and the inactive heads were incubated at the same location under the same conditions . Living and dead plant material was harvested 3 and 5 days after inoculation (dai) and further analyzed for mycotoxin levels and fungal transcriptomes. In addition to these plant-based experiments we performed a standard axenic culture experiment (subsequently termed “axenic growth”) using the resting cell method .Figure F. graminearum Ph-1 inoculated Remus wheat heads and as seen in panel A of Figure We first determined to which extent the fungal cells are able to proliferate on and in the wheat heads depending on whether there is active metabolism or not. Figure To assess, to which extent the ratio of fungal DNA to wheat DNA mirrors the activity of the fungal cells we also quantified the transcript levels of a constitutively transcribed fungal housekeeping gene relative to the transcript levels of an equivalent plant gene. Quantification of fungal and wheat GAPDH cDNA levels by qPCR Figure  gave simin vivo signals for mycotoxin induction are, they are not formed or unstable in the non-challenged plant cells. Obviously, for high induction of these SM biosynthetic genes the fungal invader needs host metabolites that are actively formed, most likely associated with the defense reaction, by the plant. Transcriptional analysis of the key biosynthetic genes and the transcription factors involved in the formation of the tested metabolites showed that the elevated mycotoxin levels detected during pathogenic growth on the living plant , butenolide and culmorin only accumulated in the living plant tissue but mycotoxin levels remained very low in the dead plant samples  which are normalized for both sequencing depth and gene length. Figure 2(FPKM) values for each condition. From this plot it becomes evident that under pathogenic and axenic conditions around 3500 genes of the F. graminearum genome are not or only rarely expressed (log2 < 1). Interestingly, around 1000 additional genes are expressed under saprophytic conditions. Considering the distribution of expression values over the genome, we saw that the majority of genes are expressed between ≥1 log2(FPKM) ≤ 5. This range covers around 9000 genes or around 2/3 of the transcribed F. graminearum genome. Due to these findings we considered from all predicted 13.826 genes the 65% most strongly expressed ones in each condition for further analysis. Using this cut off, all considered genes showed an expression level of at least log2(FPKM) ≥ 1.92 under axenic, at least log2(FPKM) ≥ 1.99 under pathogenic and at least log2(FPKM) ≥ 2.46 under saprophytic growth conditions. Relative transcript abundances in these RNA-seq data very well matched the RT-qPCR results obtained for the selected set of genes shown in Figure Total RNA was isolated from axenic as well as pathogenic and saprophytic samples harvested 3 dai and all samples were independently subjected to RNA-seq analysis. Transcript levels of genes are expressed as “FPKM” values  = 0.3] and weakly expressed under pathogenic conditions [log2(FPKM) = 1.3] but strong in saprophytic samples [log2(FPKM) = 2.7]. This is an interesting example of a gene residing within a physically-linked gene cluster but its co-regulation is condition-dependent. In the case of ORF-B the “synthetic” TRI cluster inducer L-ornithine is insufficient and the plant tissues—no matter if alive or dead—must contain a compound to generate the induction signal for ORF-B.We used L-ornithine in the axenic cultures to exclude the known trichothecene biosynthesis genes from appearing in the gene set specific for the pathogenic growth condition. Consistent with this approach we find several genes belonging to the  Figures . HoweverThis feature of non-homogenous gene expression is true also for many other SM gene clusters  or to the SM-inducing conditions (DI genes) we were able to restrict the large number of genes expressed under pathogenic conditions to genes with “active-plant” profiles possibly revealing novel virulence factors. According to our calculations 306 t Figure . As showin planta’ genes”).We summarized them in Table in planta” at different time points in wheat and barley. The most comprehensive transcriptomic study taking also previous data sets into account is available from Lysoe et al. . For the interpretation of this comparison it is important to know that the microarray-based analysis carried out by Lysoe and coworkers considered all genes which were expressed at a certain time point regardless to which level they were induced. Thus, their plant-specific gene set contained all genes which were below a certain threshold in any of the other conditions and expressed above this threshold in infected wheat, barley or in both of them. This analysis is similar with our approach shown in Figures The Venn diagrams in Figure in planta expressed” fungal genes defined in Lysoe et al.  and the tyrosinase which is part of the shikimate pathway important for SM precursors. Among the “defense-related” functions there are the two AM toxin genes and the third one, FGSG_11542 most likely encodes an aldehyde dehydrogenase involved in acetate formation. It thus may also be part of active-plant responsive SM biosynthesis as acetate can serve as a building block for several SMs. Induction of all these SM-related functions is clearly dependent on the actively defending plant and it would be worthwhile to study gene regulation of this gene set to understand the difference between the “classical” SM genes induced by the ornithine pathway also in axenic cultures and these genes which are strictly dependent on the active plant.The 31 AP genes which overlap with the previously identified F. graminearum and F. culmorum which are known in literature to reduce or even lead to loss of virulence when mutated. The trichodiene synthase-encoding gene TRI5  and PP down-regulated (TRI6) impact factor categories. The extracellular secreted lipase-encoding gene FgFGL1  but might indicate, that their transcriptional regulation does not necessarily need signals from the active, living plant to be switched on. Additionally posttranscriptional regulation might take place modifying or fine-tuning activity of the gene product. Cuomo et al. . In our experimental setup we could dissect three factors that impact on the invading fungus and vary its transcriptional response accordingly. Targeting genes that are affected by the active plant response may be a promising strategy for countermeasures against this fungal disease.In their great review Walter et al.  publisheo et al.  publisheThe most interesting category in our analysis is surely the 21 genes in the AP category which have not yet been considered in any of the previous studies. As enriched functional categories we found here disease, virulence and defense as well as detoxification, drug/toxin transport, and detoxification by export (register tab “APup_21” of Table via connection to SAM (S-adenosylmethionine) metabolism which was shown to be affected in ethionine-resistant mutants . This type of acyltransferases are known to be membrane-bound proteins that utilize long-chain fatty acid acyl-CoA and sterols as substrates to form steryl esters as precursors of ergosterol biosynthesis  or “effectors” in F. graminearum-wheat interaction.Interestingly, there are four genes of the 190 predicted SSCPs which assign to our AP_up category because they are highly induced only in the actively defending plant . Unfortunately, the corresponding peptides have not been found in the synthetic media secretome and thus, they are not confirmed SSCP-encoding genes. However, the fact that they are highly induced only in the active plant indicates that they might have a signaling or regulatory function during the So far we have mainly discussed genes up-regulated by the signals derived from the actively defending plant. But also genes specifically repressed by the active plant are interesting as they may represent possible targets of the plant defense system to keep the pathogen under control.In this category Figure  we identin planta” expressed fungal genes because the pathogenic growth status represents a mixture of genes responding to the plant tissue as substrate already present  as well as genes responding to defense signals and metabolites built up in response to the pathogenic attack. This genetic differentiation is surely relevant because it has the potential to identify novel pathogenicity factors. In addition, potentially novel plant defense targets may be present in the set of down-regulated fungal genes identified uniquely in the living plant. Our approach has led to a differentiation of genes responding to these mixed life styles and we identified 184 genes upregulated and 122 genes repressed at least four-fold by the signals the active plant generates during the infection process. We found in this gene set a substantial overlap with previously characterized “in planta” expressed genes but we identified 21 so far unrecognized genes truly responsive only to the living plant. Among them we found several uncharacterized secondary metabolite gene clusters and putative membrane proteins with signaling functions. Interestingly, most of the downregulated transcripts potentially code for proteins with unknown functions making this category highly attractive for future research. It is not unreasonable to expect that some of these novel active-plant induced or repressed genes code for novel pathogenicity factors promoting the infection process or targeting of the plant defense system. Further studies by inactivation and overexpression of these genes will probably shed new light on the F. graminearum-wheat interaction process during the pathogenic process and it will also be interesting to see which of those genes are altered in their expression profiles in the epigenetic mutants we are studying in parallel.The aim of our study was to better define the group of “F. graminearum Ph-1 wildtype  applied in this study was maintained on Fusarium minimal medium (FMM) agar plates according to standard methods  on the reproductive part between the lemma and palea of the two basal florets during anthesis (resulting in 8 * 105 spores per wheat head). Mock ears were inoculated the same way, but using water instead of spore suspension. Three ears were inoculated on the living plant representing pathogenic growth of the fungus and three ears, which were cut off the plant and shock-frozen in liquid N prior to spore application, were inoculated as “non-response” control from the wheat side representing saprophytic growth of the fungus. After inoculation the wheat heads on the living plants were covered with moistened plastic bags for the first 24 h to provide high humidity. The inoculated dead wheat heads were placed in glass petri dishes . Incubation conditions were set at 20⋅C, 50% relative humidity during daytime and 18⋅C, 50% humidity during night with a 16 h photoperiod. Harvesting was performed 3 and 5 dai by freezing the plant material in liquid nitrogen. Only palea and lemma of the inoculated florets were sampled including the respective part of the rachis. Additionally completely untreated wheat heads were sampled. After freezing in liquid nitrogen samples were stored at −80⋅C.The highly susceptible cultivar Remus . Before sample loading the grinding jars/ steel balls were baked at 270⋅C for 1 h, left at room temperature for 1 h and then pre-cooled overnight at −80⋅C. Milling was done at 30 Hz and room temperature for 20 s.Independent RNA-extractions of three distinct pathogenic and three distinct saprophytic inoculated wheat heads harvested 3 dai were delivered for RNA-Seq analysis to the VetCORE—Facility for Research .Fusarium culture supernatants were determined by applying a recently published GC-MS method  chloride [Sigma 2087272], 1 M HCl), 50 μL Griess Reagent I (N-(1-Naphthyl)ethylenediamine dihydrochloride [Sigma 33461]), and 50 μL Griess Reagent II  incubated at 37⋅C for 1 h followed by measurement of extinction at 540 nm. Culture filtrates were diluted 1: 1000 prior to the assay and as standard solutions liquid FMM media  and diluted to final nitrate concentrations of 50, 40, 30, 20, and 10 μM.In the assay 100 μL culture filtrate or standard solution were mixed with 100 μL of acidic VClIn case of the axenic minimal media cultures 1 mL of each culture filtrate was directly analyzed by liquid chromatography/electrospray ionization-tandem mass spectrometry (HPLC/ESI-MS/MS) as previously described , diluted in sterile water. The external standard curve for the EF-G assay was still done by measuring two-fold dilution series of wheat DNA starting from 100 ng/μL, but going down till 6.25 ng/μL , diluted in sterile water. For a single TaqMan reaction 2 μL template DNA was mixed with 7.5 μL Kapa Probe Fast qPCR Universal 2x Mastermix (Peqlab 07-KK4701), 4.78 μL sterile water, 0.24 μL dual-labeled probe, forward and reverse primer (each 100 pmol/ μL). Used primers and probes can be found in Brunner et al.  following the instructions of the manufacturer with one modification and one extension: Instead of eluting RNA once in 30 μL RNase-free water, elution was performed in 2 × 20 μL RNase-free water with a 3 min standing period at room temperature after each water application to the column. Additionally on-column DNase digestion was done during the extraction protocol by using RNase-Free DNase Set . RNA quantity was first checked on the NanoDrop 2000c Spectrophotometer (Thermo Scientific). Afterwards RNA quantity and integrity was validated by using the Agilent RNA 6000 Nano Kit on an Agilent 2100 Bioanalyzer machine following the instructions of the provider. The cDNA Synthesis was carried out with the RevertAid H Minus First Strand cDNA Synthesis Kit  using random hexamer primer and following the manufacturer's protocol. Details on procedure and conditions for cDNA synthesis and determination of infection rates are given in Datasheet F. graminearum and FGDB annotation version 3.2 was retrieved from http://www.helmholtz-muenchen.de/en/ibis/institute/groups/fungal-microbial-genomics/resources/index.html  values between the specific contrasts, also indicated by square brackets, were calculated like for AP: patho vs. sapro & axo; PP: sapro & patho vs. axo; and DI: axo & patho vs. sapro.Factor dependent transcription was calculated as follows: Factor AP was calculated according to formula AP = [patho] − [(sapro + axo)/2], where patho, sapro, and axo represent log2(FPKM) values of the experiments pathogenic- saprophytic- and axenic- growth. Similarly, PP was calculated as PP = [(sapro + patho)/2] − [axo] and DI = [(axo + patho)/2] − [sapro]. Means and standard deviation were calculated by first order Taylor expansion in R. To determine if a gene is affected by a single factor t-tests as described above.The reasoning behind formula AP = [patho] − [(sapro + axo)/2]: patho is the only condition where a living plant is present so any difference between patho and the combination of sapro and axo is likely down to active plant action. PP = [(sapro + patho)/2] − [axo]: in both experiments sapro and patho is plant material but not in axo, so a deviation from zero may be cause by the plant. DI = [(axo + patho)/2] − [sapro]: both axo and patho experimental setups provide limited supply with nutrients vs. in sapro setup strong growth was observed what hints toward satisfactory supply. To determine if a factor is singly/mainly responsible we performed SB, IM, ML, and JS planned and performed fungal and plant experiments. HB, SB, CS, MM, UG, and JS analyzed and interpreted transcriptome data. BW, MS, and RS analyzed and interpreted metabolomic data. SB, HB, and JS wrote the main parts of the manuscript with contribution from all other authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


ATP) channel activity, we tested the hypothesis that HDIs affect cardiac KATP channel subunit expression. Two different HDIs, romidepsin and trichostatin A, caused ~20-fold increase in SUR2 (Abcc9) subunit mRNA expression in HL-1 cardiomyocytes. The effect was specific for the SUR2 subunit as neither compound causes a marked change in SUR1 (Abcc8) expression. Moreover, the effect was cell specific as neither HDI markedly altered KATP subunit expression in MIN6 pancreatic β-cells. We observe significant enrichment of the H3K9Ac histone mark specifically at the SUR2 promoter consistent with the conclusion that chromatin remodeling at this locus plays a role in increasing SUR2 gene expression. Unexpectedly, however, we also discovered that HDI-dependent depletion of cellular cholesterol is required for the observed effects on SUR2 expression. Taken together, the data in the present study demonstrate that KATP subunit expression can be epigenetically regulated in cardiomyocytes, defines a role for cholesterol homeostasis in mediating epigenetic regulation and suggests a potential molecular basis for the cardiac effects of the HDIs.Histone deacetylase inhibitors (HDIs) are under investigation for the treatment of a number of human health problems. HDIs have proven therapeutic value in refractory cases of cutaneous T-cell lymphoma. Electrocardiographic ST segment morphological changes associated with HDIs were observed during development. Because ST segment morphology is typically linked to changes in ATP sensitive potassium (K Histone deacetylase inhibitors (HDIs) have emerged as potential therapeutic agents for the treatment of cancer and heart disease  channels during ischemia  and four sulfonylurea receptor (SUR1 or SUR2) subunits that are encoded by four separate genes   for monitoring reaction progress. Twenty nano gram of template cDNA was used in all reactions. Reactions with each primer/probe pair and template were performed in triplicate. Relative mRNA expression (compared to Hprt) is reported as 2−ΔCt * 1000 for untreated HL-1 and MIN6 cells. The fold change in expression in response to drug treatment is reported as 2−ΔΔCt, where ΔΔCt = ΔCt (drug)−ΔCt (notreatment).Relative expression of KCells were washed with PBS and disrupted with RIPA lysis buffer (Santa Cruz) supplemented with protease inhibitors (Roche). Protein concentration was determined by the BCA method (Pierce). For Western analysis, proteins  were separated by PAGE and transferred to PVDF membrane. Signal was visualized with the SuperSignal West Dura ECL substrate (Pierce).8 cells were incubated with 1% formaldehyde in 1X PBS for 10 min at room temperature followed by quenching with 0.125 M glycine for 5 min. After cell lysis, cross-linked chromatin was digested with micrococcal nuclease, followed by sonication to yield fragments of 200–1000 bp. Soluble chromatin fragments were incubated with antibodies against AcH3 , control IgG  and H3 . Bound chromatin was precipitated using MagnaChip protein A+G magnetic beads (Millipore). Precipitated complexes were eluted, decrosslinked and purified. Purified DNA was amplified by qPCR with primer set corresponding to the promoter regions (−1 kB) for SUR1 and SUR2 (Qaigen). The data was analyzed according to comparative Ct method, normalized to IgG control and reported as fold change in binding relative to control.Chromatin immunoprecipitation (ChIP) assay was performed using the SimpleChip enzymatic chromatin IP kit  according to manufacturer's protocol. Briefly, 1 × 10MouseRef-8 v2.0 Expression BeadChips  were used to measure relative levels of mRNA expression for 24,854 well-established annotated coding transcripts. Preparation of labeled cDNA, hybridization, imaging, and data collection were carried out at the Cleveland Clinic Genome Core Facility. Background subtracted, quantile normalized data were generated and analyzed using GenomeStudio , GenePattern 2.0  and GATHER  if the effects are tissue specific.The objective of the present study was to test the hypothesis that histone deacetylase inhibitors (HDIs) cause changes in KATP subunit expression and that this occurs in a cell or tissue specific manner.Figure To begin to elucidate the mechanism underlying the TSA-dependent increase in SUR2 expression, we confirmed that the HDAC inhibitors induced a global increase in histone acetylation in HL-1 cells. As expected, treatment with TSA significantly increased the amount of acetylated histone H3 and H4 without changes in the total histone protein Figure . MoreoveTnni2) was expressed at very low levels and the other (Ttn) was only decreeased 2.8-fold. We also annotated the 171 common gene dataset for enrichment of transcription factor binding sites (TRANSFAC v 8.2). Interestingly, this analysis demonstrated significant enrichment for sterol response element binding proteins (SREBPs) activity in response to HDIs. . The enrichment of SREBP binding sites in the subset of transcripts affected by HDIs suggests that SREBP may play a role in mediating the effects of HDIs on KATP channel subunit expression. This possibility was wholly unexpected, however, it should be pointed out that a previous study noted the presence of SREBP binding sites in the promoter regions of the SUR2 and Kir6.2 genes (Philip-Couderc et al., To gain further mechanistic insight, we next performed a comprehensive genome-wide expression BeadArray profiling to determine the transcripts that are up- and down-regulated by HDIs in HL-1 cells Figure . Three hA major physiological regulator of SREBP activity is cellular cholesterol. Reduced cholesterol levels in the endoplasmic reticulum trigger a sequence of events leading to cleavage of full length SREBP, allowing translocation of the transcriptionally active SREBP cleavage product to the nucleus (Jeon and Osborne, ATP expression, we assessed SUR2 expression in HL-1 cells incubated in culture medium supplemented with increasing concentrations of cholesterol and a constant concentration of TSA. Addition of cholesterol to the media completely restored cellular cholesterol as evidenced by the restoration of filipin staining (Figure ATP subunit expression in HL-1 cardiomyocytes.To determine whether TSA-dependent disruption of cholesterol homeostasis mediates the effects on Kg Figure . Cholestg Figure . ImportaATP channel subunits. First, we show that inhibiting HDAC activity specifically increases SUR2 gene transcription in HL-1 cardiomyocytes. This result implies that under normal circumstances HDACs are recruited to the SUR2 gene locus in HL-1 cells contributing to the suppression of SUR2 expression in these cells under basal conditions. Second, we show that disruption of cellular cholesterol homeostasis is a critical mediator of HDI effects in HL-1 cells. This result implies that cholesterol homeostasis, in general, may be an important determinant of KATP expression in the cardiovascular system.The data in the present study demonstrate two novel aspects regarding the transcriptional control of KATP channel subunit expression providing evidence that KATP channel subunit expression is normally determined in part by HDAC-dependent epigenetic regulation.Covalent histone modifications including acetylation, methylation, ubiquitylation, sumoylation, and phosphorylation are increasingly recognized as epigenetic regulators of when and where genes are actively expressed (Kouzarides, ATP channel expression. First, HDIs exhibit cell specific effects as evidenced by the observation that KATP subunit expression is markedly affected in HL-1 cardiomyocytes but unaffected in MIN6 cells. HL-1 cells appear to be primed to express SUR2, but tonic suppression by HDACs normally maintains a low level of SUR2 expression. In contrast, the appropriate transcriptional activators of SUR2 expression appear to be absent in MIN6 cells and in this case suppression of SUR2 gene expression by HDACs would be redundant and is therefore not present. In addition to cell specificity, epigenetic regulation of KATP channel expression is also subunit specific. HDIs specifically increase histone acetylation at the SUR2 gene but have no effect at the SUR1 locus. This demonstrates that HDACs are recruited specifically to the SUR2 locus in order to establish the pattern of KATP subunit expression observed in HL-1 cells.Specificity is a key feature of epigenetic regulation of KATP channel composition or function has been shown to cause arrhythmias or abnormal ECG responses to ischemia. Using transgenic models that overexpress KATP subunits, we have shown that disrupting normal KATP composition by overexpressing SUR1 is arrhythmogenic (Flagg et al., ATP, for example, completely abolishes the ST segment elevation associated with ischemia (Kubota et al., ATP channel openers such as pinacidil have been shown to induce ST segment elevation independent of ischemia (Yan and Antzelevitch, ATP is a particularly potent means of modulating cardiac action potential duration. Activation of just 1% of the total KATP channels available has been shown to shorten the APD by 50% (Nichols et al., ATP could be manifest as changes in the ECG.Disruption of the appropriate KATP channels (Kubota et al., ATP channel subunit expression provide a potential molecular basis for the cardiac effects associated with HDIs. Similarly, re-entrant arrhythmias can be promoted by activation of KATP (Billman, ATP channel expression may also contribute to the electrophysiological events observed during HDI-therapy.The onset of cardiac arrhythmias has been observed in several clinical and pre-clinical studies of HDI therapy (Berry et al., Billman, . Other mBillman,  or non-s−−∕−− mice (Kim et al., The second major finding in this study is the discovery of a key role for cholesterol depletion in mediating the effect of HDIs on SUR2 expression. The key clue that focused attention on cholesterol was the association of SREBP with the gene changes induced by the HDIs Figure . SREBPs ATP subunit expression. This finding suggests the very intriguing conclusion that there is a link between cellular cholesterol homeostasis and KATP expression. There have been no previous studies that directly link cholesterol homeostasis with KATP channel expression. In silico analysis previously identified SREBP binding sites in the SUR2 and Kir6.2 promoters (Philip-Couderc et al., ATP expression. Hypercholesterolemia, which is expected to inhibit SREBP, has been associated with impaired ischemic preconditioning (Csonka et al., ATP channels (Suzuki et al., ATP subunit expression with consequent arrhythmias.The data indicate that maintaining cellular cholesterol prevents the effect of HDIs on KATP channel subunits. This defines a role for HDACs in determining which KATP channel subunits are expressed in cardiomyocytes but not pancreatic β-cells. Moreover, the data demonstrate a key role for cholesterol homeostasis in regulating this process. Taken together, these novel observations could lead to new strategies to modulate sarcolemmal KATP channel structure and function in the heart where it protects against stress and ischemia (Flagg et al., In summary, the results presented in this study demonstrate that HDIs such as TSA and romidepsin cause marked changes in the expression of KThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Stromal cells with a myofibroblast phenotype present in the normal human esophagus are increased in individuals with gastro-esophageal reflux disease (GERD). We have previously demonstrated that myofibroblasts stimulated with acid and TLR4 agonists increase IL-6 and IL-8 secretion using primary cultures of myofibroblasts established from normal human esophagus. While primary cultures have the advantage of reflecting the in vivo environment, a short life span and unavoidable heterogeneity limits the usefulness of this model in larger scale in vitro cellular signaling studies. The major aim of this paper therefore was to generate a human esophageal myofibroblast line with an extended lifespan. In the work presented here we have generated and characterized an immortalized human esophageal myofibroblast line by transfection with a commercially available GFP-hTERT lentivirus. Immortalized human esophageal myofibroblasts demonstrate phenotypic, genotypic and functional similarity to primary cultures of esophageal myofibroblasts we have previously described. We found that immortalized esophageal myofibroblasts retain myofibroblast spindle-shaped morphology at low and high confluence beyond passage 80, and express α-SMA, vimentin, and CD90 myofibroblast markers. Immortalized human esophageal myofibroblasts also express the putative acid receptor TRPV1 and TLR4 and retain the functional capacity to respond to stimuli encountered in GERD with secretion of IL-6. Finally, immortalized human esophageal myofibroblasts also support the stratified growth of squamous esophageal epithelial cells in 3D organotypic cultures. This newly characterized immortalized human esophageal myofibroblast cell line can be used in future cellular signaling and co-culture studies. Gastro-esophageal reflux disease (GERD) is one of the most common causes of chronic esophageal injury. Mucosal injury stems from refluxed gastric acid, pepsin and bile along wiPrimary esophageal myofibroblasts grown on plastic typically either senesce or become transformed within 25 passages. The recent advances in our understanding of the potential role for esophageal myofibroblasts in the pathogenesis of GERD injury  requiresThe major aim of this paper was to generate a human esophageal myofibroblast line with an extended lifespan to facilitate future investigation of the mechanisms by which human esophageal myofibroblasts respond to stimuli encountered in esophageal inflammatory disorders such as GERD (e.g. acid and TLR4 ligands) in the complex environment of the esophageal mucosa. Based on literature review ,8 we choPrimary cultures of esophageal myofibroblasts  were est4 cells/well) were treated with pH 4.5 serum-free myofibroblast media for 15 minutes followed by replacement with fresh serum-free myofibroblast media and collection of media at 6 hours. Cells were treated with 10 μg/ml LPS for 24 hours and conditioned media was collected. IL-6 ELISA was performed according to manufacturer’s instructions .Primary esophageal myofibroblasts and GFP-hTERT transfected esophageal myofibroblasts were cultured in myofibroblast media as previously described and incubated in 37°C with 5% CO2. Treatment with acidified media and LPS was performed as previously described [Flow cytometry was performed to further characterize hTERT transfected cells. Antibodies to CD90 conjugated to APC, CD45 conjugated to PE-Cy7, CD31 conjugated to eFluor450, and CD324 conjugated to PerCP710 were purchased from eBioscience . Briefly, single- and multi-color immunostaining was performed according to standard surface FACS staining protocols (eBioscience). Cells were analyzed by flow cytometry using LSRII cytometer  according to the manufacturer’s protocol. Forward and side scatters thresholds were used to exclude debris and discriminate single live cells. Viability was also assayed using Annexin V and 7-aminoactinomycin D  and an additional gate was used to exclude dead cells from the analysis. Flow cytometry data were analyzed with FlowJo software . Appropriate isotype controls were used to determine nonspecific antibody staining.Immunocytochemistry for α-SMA, vimentin, TLR4  and TRPV1  were performed on transfected myofibroblasts grown in chamber slides at several passages as previously described . Cells wRNA was harvested from transfected myofibroblasts at several passages using the GeneElute MammalianTotal RNA Mini-prep Kit (Sigma-Aldrich), per manufacturer’s instructions.cDNA was synthesized and mRNA expression of the following genes was evaluated: α-SMA , vimentin , hTERT , TRPV1 , TLR4 . Human 18s  was used as the endogenous control to normalize samples using the ΔΔCT method of relative quantitation, where CT is the threshold cycle. Expression data is presented in log form. Primary esophageal myofibroblasts were used as controls.Human esophageal epithelial cell line EPC2  were cultured in a 3D air-liquid interface (ALI) organotyic culture (OTC) system utilizing primary and GFP-hTERT esophageal myofibroblasts in the collagen matrix support layer. Culture conditions were as per published methods  with thet-test .All experiments were performed in triplicate and data presented as means ± SE. Data were analyzed using Student’s two-tailed type 2 hTERT and eGFP. hTERT is driven by a CMV promoter and GFP is driven by a separate EF1a promoter. Flow cytometry followed by FACS was performed on transfected cells to sort out GFP expressing cells. Up to 0.1% of cells expressed GFP. GFP expressing cells were cultured and evaluated for hTERT expression by quantitative RT-PCR. Elevated hTERT transcript levels were detected in GFP-hTERT transfected human esophageal myofibroblasts .We generated immortal esophageal myofibroblasts by transfecting previously characterized early passage primary esophageal myofibroblasts established from normal human esophagus  with a cFig 2). Similar to primary cultures, transfected cells at low passage demonstrated spindle-shaped morphology at low and high confluence. Transfected esophageal myofibroblasts examined up to passage 89 also maintained myofibroblast morphology at low and high confluence and did not senesce, consistent with immortalization. Overall, immortalized esophageal myofibroblasts did not demonstrate identifiable changes in morphology between early and later passages, remained dependent on tissue-culture treated plates and grew in a monolayer. They did not grow in 0.1% serum and required myofibroblast-media used for primary cells for growth. Telomerase expressing human esophageal myofibroblasts remained dependent on solid support for growth.We then evaluated the morphology of cultured GFP-hTERT transfected cells by examination with an inverted microscope. Morphology of transfected myofibroblasts was similar to that of primary cultures from which they were established . Protein expression of these cytoskeletal proteins was readily detected in immortalized MFs  and was similar to that previously described for primary myofibroblasts [Fig 3D).SMA and vimentin mRNA expression was variable across myofibroblast primary cultures as previously reported and easily detected in hTERT transfected immortalized MFs .We then evaluated expression of two receptors for injurious luminal and epithelial derived signals encountered in GERD in transfected cells. We have previously shown that primary cultures of esophageal myofibroblasts express transient receptor potential channel vanilloid subfamily 1 (TRPV1) and TLR4. TRPV1 is the putative acid receptor and TLR4 is responsible for signaling in response to gram negative bacteria and DAMPs . TRPV1 aFig 5). A more profound increase in IL-6 secretion in response to LPS in comparison to treatment with acidified media was observed in immortalized esophageal myofibroblasts. This pattern has been previously reported in primary esophageal myofibroblasts [We had previously reported that primary esophageal myofibroblasts increase secretion of IL-6 in response to pH 4.5 acidified media and TLR4 ligand LPS. We therefore evaluated the response of immortalized human esophageal myofibroblasts cells to treatment with pH 4.5 acidifed media and TLR4 ligand lipopolysaccharide. Similar to primary esophageal myofibroblasts, immortalized cells increase IL-6 secretion  in response to acid and LPS  .We further investigated the functional similarity between primary and immortalized esophageal myofibroblasts in 3D organotypic culture (OTC) that recapitulates stratified squamous epithelium of the esophagus. Immortalized human esophageal cells cultured in collagen constricted the collagen matrix to a similar degree as primary esophageal myofibroblasts .We then showed that immortalized esophageal myofibroblasts support the stratified growth of human esophageal squamous epithelial cells EPC2  similar to primary esophageal myofibroblasts (Although various approaches to immortalization have been described, ectopic expression of hTERT is a recognized method of overcoming or delaying the senescence that limits the long term use of primary cells ,10. SuccWe found that immortalized esophageal myofibroblasts retain myofibroblast spindle-shaped morphology at low and high confluence, express α-SMA and vimentin myofibroblast markers, express stromal cell surface protein CD90, and lack expression of hematopoietic, endothelial, and epithelial markers. Immortalized esophageal myofibroblasts also express TRPV1 and TLR4 and retain the functional capacity to respond to stimuli encountered in GERD with secretion of IL-6. Immunostaining demonstrates similar diffuse cytoplasmic expression of these receptors in both primary cultures and immortalized esophageal myofibroblasts. Cell surface expression and the mechanisms by which stimulants gain entrance into the cell are topics of ongoing investigation. Similar to primary cultures, immortalized esophageal myofibroblasts also support the growth of epithelial cells in 3D OTC.The GFP component of immortalized esophageal myofibroblasts we have generated will allow for effective identification of human esophageal myofibroblasts in multi-cellular and co-culture studies. Immortalization allows for the ability to proceed with cell to cell signaling studies in co-culture or 3D OTC format in a reproducible fashion. Such studies are required to fully delineate the cellular signaling mechanisms mediating complications of GERD related esophageal inflammatory disorders such esophagitis, strictures, intestinal metaplasia and esophageal adenocarcinoma.