Current therapeutics are only able to slow disease progression, therefore there is a need to explore alternative, novel treatment options. There is increasing evidence that the 3′, 5′ cyclic adenosine monophosphate (cAMP) pathway is an important modulator in the development of fibrosis, with increasing levels of cAMP able to inhibit cellular processes associated with IPF. In this study we investigate the expression of Gs-coupled GPCR. The function of selected agonists at expressed receptors was then tested in a cAMP assay, and for their ability to inhibit fibroblast proliferation and differentiation.Using TaqMan arrays we determined that fibroblasts express a range of G2 (PGE2) receptor 2 and 4, melanocortin-1, β2 adrenoceptor, adenosine 2B, dopamine-1, and adenosine 2A receptors were expressed in HLF. Measuring cAMP accumulation in the presence of selected Gs-coupled receptor ligands as well as an adenylyl cyclase activator and inhibitors of phosphodiesterase showed formoterol, PGE2, treprostinil and forskolin elicited maximal cAMP responses. The agonists that fully inhibited both fibroblast proliferation and differentiation, BAY60–6583 and MRE-269, were partial agonists in the cAMP accumulation assay.Expression analysis of GPCR showed that the prostacyclin, prostaglandin EIn this study we identified a number of ligands that act at a range of GPCR that increase cAMP and inhibit fibroblast proliferation and differentiation, suggesting that they may provide novel targets to develop new IPF treatments. From these results it appears that although the cAMP response is important in driving the anti-fibrotic effects we have observed, the magnitude of the acute cAMP response is not a good predictor of the extent of the inhibitory effect. This highlights the importance of monitoring the kinetics and localisation of intracellular signals, as well as multiple pathways when profiling novel compounds, as population second messenger assays may not always predict phenotypic outcomes.The online version of this article (10.1186/s12931-018-0759-2) contains supplementary material, which is available to authorized users. Idiopathic pulmonary fibrosis (IPF) is an age-related, chronic and progressive fibrotic lung disease, with an annual incidence in the UK of 7.44 per 100,000 people, and a median survival of approximately 3 years from diagnosis . An incrA key therapeutic goal in the treatment of IPF is to reduce tissue fibrosis, which may be achieved by enhancing signals that counteract fibrotic processes, and/or inhibit signals and mediators that promote fibrosis. There are at present two drugs that are now approved for use in the treatment of IPF; nintedanib and pirfenidone. Nintedanib inhibits multiple receptor tyrosine kinases (RTK)  and pirfs-coupled G protein-coupled receptors (GPCR) thus activating adenylyl cyclase (AC) and increasing levels of cAMP, inhibit lung fibroblast migration, proliferation and differentiation. For example, prostaglandin E2 (PGE2) acting at PGE2 receptor 2 (EP2) and 4 (EP4) and iloprost acting at the prostacyclin (IP) receptor, were shown to have an inhibitory effect on proliferation and differentiation of lung fibroblasts, via cAMP accumulation and protein kinase A (PKA) activation [As RTK predominantly signal through the Ras/mitogen-activated protein kinase (MAPK) signalling cascade, it is likely that this pathway is responsible for many of the pro-fibrotic responses observed in IPF . There itivation –16. Additivation . Furthertivation , 19.s-coupled GPCR in order to identify potential new targets to treat IPF. We have explored the ability of Gs-coupled GPCR present in HLF to increase global cAMP using high-throughput compatible, homogenous assay kits. We have further explored how the observed increases in cAMP accumulation translate into an ability to inhibit fibroblast proliferation and differentiation, in order to determine whether any of the selected GPCR would be suitable for exploring further as novel targets to treat IPF.The present study was performed in order to evaluate the anti-proliferative capacity of a range of GFoetal bovine serum (FBS), trypsin-EDTA, Hoechst 33,342, RNAqueous kit, TURBO DNase and High Capacity RNA-to-cDNA kit were purchased from Life Technologies . CGS-21680, BMY-45778, BAY60–6583, iloprost, and NECA were purchased from Tocris . Beraprost was bought from Cayman Chemical Company . PDGF-BB was purchased from R&D Systems . MRE-269 (CAS:475085–57-5) and AGN-205204 (CAS:802906–77-0) were synthesized in house at Novartis . Phospho-p44/42 ERK1/2 (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb primary antibody was purchased from Cell Signalling Technology . CF™647-conjugated Affini-pure goat anti-rabbit IgG and CF™647-conjugated Affini-pure goat anti-mouse IgG were purchased from Biotium, Inc., . Pierce™ LDH Cytotoxicity Assay Kit and CellEvent™ Caspase-3/7 Red Detection Reagent were purchased from ThermoFisher . Staurosporine was purchased from VWR International Ltd. . 96-well white ViewPlates, AlphaScreen cAMP and DELFIA proliferations assay kits were purchased from Perkin Elmer Life Sciences , 96-well black ViewPlates were purchased from Corning , and 384-well black, μ-clear Viewplates were purchased from Greiner bio-one . HitHunter™ cAMP assay kits were purchased from DiscoverX . All other reagents were purchased from Sigma Aldrich .− 1 D-glucose, L-glutamine, pyruvate, FBS (10% v/v), and 25 mM HEPES at 37°C, 5% CO2, in a humidified atmosphere. For experiments cells were harvested using trypsin/EDTA and seeded in their sub-culture medium. Two HLF donors were used in the functional studies: HLF d91019001.2 and HLF 9017402.1, both from healthy/non-smoker Caucasian donors.All studies were performed on normal human lung fibroblasts (C-12361), which were purchased from Promocell . HLF were maintained in DMEM supplemented with 4.5 g LT) set at 0.2 for all samples.Expression of GPCR was determined in HLF using high density, 384-well GPCR TaqMan arrays as previously described . RNA fro80 concentration of PDGF (3 ng/mL) for 10 min, at 37°C, 5% CO2. After stimulation, cells were fixed in 4% paraformaldehyde, washed 3× in PBS (with Ca2+/Mg2+), and incubated with permeabilising blocking buffer (dPBS (with Ca2+/Mg2+), 10% FBS (v/v), 0.1% Triton X-100 (v/v)) for 1 h at 37°C. Wells were then washed 3× in wash buffer , 0.05% (v/v) Tween-20) and incubated with rabbit anti-pERK1/2 antibody , and FITC-conjugated Affini-pure goat anti-rabbit IgG (1:1000 dilution in blocking buffer) for 30 min at 37°C. Fluorescence was then quantified on the InCell2000, using DAPI settings to visualize nuclei (0.1 s exp) and FITC settings for pERK (2 s exp).HLF were seeded overnight at 6000 cells/well in 96-well black clear bottom plates then starved for 24 h in the growth medium devoid of all additives. To assess ERK phosphorylation (pERK), cells were incubated in growth medium devoid of all additives  for 30 min with a range of concentrations of forskolin. After this time, cells were stimulated with an EC80 PDGF response.To quantify levels of pERK fluorescence, a standard Multi-Target Analysis algorithm was used in the INCell Analyzer Workstation (v3.7.1). To account for the inter-assay variation in levels of ERK phosphorylation produced in each experiment, data were normalized to the ECw/v HSA, and 5 μM rolipram. Rolipram was included in these assays to inhibit PDE activity, which resulted in increased maximal responses of cAMP accumulation, without any effect on EC50 values generated (data not shown). Rolipram was excluded when measuring cAMP levels in response to PDE inhibitors. cAMP levels were measured using either HitHunter cAMP assay or AlphaScreen competition assay following manufacturer protocol, and were assessed on either a BMG LABTEK ClarioStar or a Packard EnVision plate reader. cAMP concentrations in each well were determined using a standard curve. To account for the inter-assay variation in levels of cAMP produced in each experiment, data were normalized to the maximal forskolin response.HLF were grown to confluency in 96-well white ViewPlates, before incubation with a range of agonist concentrations, or vehicle, for 2 h at room temperature in HBSS with 5 mM HEPES, 0.1% w/v HSA, for 24 h.Proliferation of HLF was measured by incorporation of bromodeoxyuridine (BrdU). HLF were seeded overnight at 4000 cells/well in 96-well black ViewPlates, before being starved for 24 h in culture medium devoid of FBS. Proliferation was measured after incubation with a range of concentrations of FBS or PDGF, in DMEM supplemented with 0.1% For anti-remodelling assays using BrdU incorporation, cells were incubated for 24 h with a range of concentrations of test compounds in the presence of near maximal concentrations of FBS or PDGF in DMEM supplemented with 0.1% w/v HSA. Proliferation was assessed using the DELFIA BrdU incorporation assay kit following manufacturer protocol. Fluorescence was measured using a Packard EnVision plate reader. To account for the inter-assay variation in levels of proliferation in each experiment, data were normalized to the PDGF or FBS response.To assess the differentiation of fibroblasts to the myofibroblast phenotype, immunofluorescence was used to monitor increases in α smooth muscle actin (αSMA). HLF were seeded overnight at 1000 cells/well in 384-well black clear bottom plates, before being starved for 24 h growth medium devoid of FBS.2 in the presence of a near maximal concentration of TGFβ (0.3 ng/mL), to promote differentiation in in DMEM supplemented with 0.1% w/v HSA. Following stimulation, cells were fixed in 4% paraformaldehyde, washed 3× in PBS (with Ca2+/Mg2+), and incubated with permeabilising blocking buffer (dPBS (with Ca2+/Mg2+), 10% FBS (v/v), 0.1% Triton X-100 (v/v)) for 1 h at 37°C. Wells were then washed 3× in wash buffer , 0.05% (v/v) Tween-20) and incubated with mouse anti-α SMA (1:1000 dilution in permeabilising blocking buffer) for 1 h at room temperature, with gentle shaking. Following 3× wash in wash buffer, wells were incubated with blocking buffer containing Hoechst to stain for nuclear DNA (2 μM), and FITC-conjugated Affini-pure goat anti-rabbit IgG (1:1000 dilution in blocking buffer) for 30 min at 37°C. Fluorescence was then quantified on a widefield ImageXpress Micro microscope, with a Plan Fluor 20X objective, DAPI filter cube , 50 ms exposure time for nuclei, and FITC filter cube , 200 ms exposure time for αSMA.For anti-remodelling assays using FMT, cells were incubated for 48 h with a range of concentrations of test compounds and incubated for 48 h, at 37°C, 5% COTo quantify increases in αSMA fluorescence, a standard Multi-Wavelength Cell Scoring algorithm was used in MetaXpress 5.3 software . To account for the inter-assay variation in levels of αSMA expression produced in each experiment, data were normalized to the response to TGFβ.Data were analysed using GraphPad Prism (Version 7.00) and results expressed as mean ± standard error of mean (SEM), unless otherwise stated. Concentration-response data were fitted using a four-parameter logistic equation. Expression of GPCR mRNA is reported as 2 TO - ΔCT compared to the mean Cycle Threshold (CT) of the housekeeping genes and each value is the mean ± SD of 4 biological replicates. The Pearson correlation coefficient (r) was determined using the standard correlation function in GraphPad prism, followed by a two-tailed T test to determine significance.We first aimed to determine whether cAMP accumulation leads to inhibition of the pERK signal cascade in HLF, which may in turn suggest a role for cAMP in the inhibition of proliferation. Inhibition of ERK phosphorylation by the MEK inhibitor PD0325901 blocked PDGF-mediated proliferation of HLF. In turn forskolin, which increases cAMP through direct activation of AC, inhibited PDGF-mediated ERK phosphorylation , β2 adrenoceptor, adenosine 2B (A2B), EP4, dopamine-1, and adenosine 2A (A2A)  that relates to near maximal proliferation  .IPF is driven by a number of pro-fibrotic processes, therefore we next aimed to determine if the same agonists were also capable of inhibiting another key component of fibrosis, TGFβ-induced FMT. TGFβ is a key driver of fibrosis and was able to robustly promote FMT in HLF with ECIn order to test the ability of compounds to inhibit FMT, HLF were treated for 48 h with a range of concentrations of the selected ligands in the presence of a TGFβ concentration (0.3 ng/mL) that relates to near maximal FMT. Agonists and PDE inhibitors inhibited TGFβ-driven FMT with varying levels of efficacy and potency Fig. , however2 = 0.06, p = 0.41), or TGFβ-mediated FMT . This was most apparent with formoterol and treprostinil, which despite generating high levels of cAMP were relatively ineffective at inhibiting proliferation and differentiation. In contrast, BAY60–6583 and MRE-269 exhibited higher efficacy for the inhibition of proliferation and FMT than cAMP accumulation.Because the assays like the simple, homogenous second messenger system used here are often utilised in high throughput screening to identify novel agonists for cAMP accumulation, we wanted to explore the relevance of this method for predicting the inhibition of HLF proliferation and differentiation. To do this, we generated correlation plots for maximal levels of cAMP generation against the maximal inhibition of proliferation or FMT Fig. . There wIPF is a devastating disease, and with current therapeutics only able to slow disease progression and being associated with severe side effects, further research in IPF aetiology and drug discovery is urgently required. A key feature of IPF is airway remodelling which is driven by fibroblast proliferation and differentiation. This leads to the formation of fibroblast and myofibroblast foci that excrete excessive amounts of extracellular matrix resulting in airway stiffening and loss of pulmonary function. It is this airway remodelling that is associated with disease severity , 22 suggs-coupled GPCR in HLF, as well as their ability to increase cAMP and subsequently inhibit HLF proliferation and differentiation in order to identify new targets to treat IPF that will complement existing therapies.Approved therapies and novel approaches undergoing clinical trials in IPF take the approach of inhibiting a range of pro-fibrotic stimuli, often GF-RTK combinations that predominantly signal via the MAPK pathway. Using fibroblast proliferation to model aspects of lung fibrosis, we demonstrated that inhibition of the MAPK pathway completed blocked PDGF-mediated proliferation, and that ERK phosphorylation could be completely inhibited by increasing global cAMP. For this reason, we wanted to explore the expression of Gs-coupled GPCR in fibroblasts, including related receptor families such as those for prostaglandins  and adenosine (A2A and A2B), as well as the MC1, β2 adrenoceptor and dopamine-1 receptors. Importantly, not all of these receptors resulted in functional cAMP responses, as measured by high throughput, homogenous assay systems. This highlights the need to look not only at mRNA levels, but protein expression and function as well to confirm active receptors are present.We observed mRNA expression of a number of different GTo model chronic aspects of IPF we chose to study HLF proliferation and differentiation, as HLF are key mediators of fibrosis and contain human receptors and signalling proteins at relevant levels. Proliferation was monitored using two complimentary methods, BrdU incorporation into newly synthesized DNA of actively proliferating cells, and direct cell counting. Differentiation was monitored using an FMT assay that measures the increased expression of αSMA that indicates a myofibroblast phenotype. Using the BrdU incorporation assay we were able to quantify proliferation in response to both PDGF and serum (which contains multiple growth factors and proliferative stimuli) at an earlier stage compared to the cell counting method. This is likely due to the different sensitivities of the assays as well as the time course of responses being measured. We also observed that full concentration-response curves for the inhibition of cell counting could not be obtained for all ligands. For this reason, we focused on the BrdU incorporation and FMT assays to explore the responses further.2, EP2, EP4, and A2B receptors, as well as forskolin, an activator of AC, were able to robustly inhibit HLF proliferation and differentiation. Interestingly, some agonists such as MRE-269, AGN205204 and BAY 60–6583 were able to fully inhibit either proliferation or FMT, despite being partial agonists for cAMP accumulation. This demonstrates that many of the agonists we have tested in this study have the potential to inhibit multiple processes that contribute to IPF clinically. It is important to note however that these studies were performed on fibroblasts from healthy donors, and therefore these results should be confirmed in fibroblasts from IPF patients, which may differ in their established phenotype.Using these models, we have demonstrated that selected agonists for the IP, βWe were able to demonstrate using bother fibroblast proliferation and differentiation that cAMP accumulation does not correlate with the degree of inhibition of chronic phenotypic responses. This highlights the importance of developing novel treatments for IPF on the basis of their chronic effects rather than acute cAMP responses. For example, although iloprost has been described as a potential treatment for IPF due to its protective effects against bleomycin-induced pulmonary fibrosis in mice , our dat2 has been shown to inhibit cellular markers of fibrosis [2 signalling in lung fibrosis [2 &/or EP4 agonists are strong candidates for novel IPF treatments. Recent studies have also identified the A2B receptor as an important target in the regulation of both acute and chronic lung disease, with agonists being considered for the treatment of acute lung injury and antagonists being considered for the treatment of chronic lung diseases [2B agonist BAY 60–6583, although not very potent, showed robust inhibition of HLF proliferation.A number of the GPCR we identified here have previously been implicated in the treatment of IPF or other chronic lung diseases. The endogenous EP receptor agonist PGEfibrosis , 16, andfibrosis . This cofibrosis , and thediseases –28. In a2 adrenoceptor agonists are widely used for the treatment of lung diseases such as chronic obstructive pulmonary disease (COPD), providing relief by inducing bronchodilation of airway smooth muscle. We have shown here that the long acting β2 adrenoceptor agonists’ salmeterol and indacaterol may also act as anti-fibrotic agents in IPF through the robust inhibition of HLF proliferation and differentiation.Currently, inhaled βAlthough we did not observe any acute cAMP accumulation with the PDE inhibitor IBMX, we did observe some inhibition of both proliferation and FMT at higher concentrations. In the isolated fibroblasts there may not be sufficient basal cAMP turnover in order for PDE inhibitors to substantially increase levels of cAMP during the shorter cAMP assay, but with the extended incubation for the phenotypic assays (24 or 48 h), IBMX may be able to cause some accumulation of cAMP that is driving this inhibitory effect. In support of this, PDE inhibitor use is being explored for IPF and they have been shown to be effective in the bleomycin model , 19. It The mechanisms behind the apparent disconnect we observed between acute cAMP accumulation and inhibition of chronic phenotypic responses is not yet clear, however there are a number of factors that need to be considered. Firstly, proliferation and differentiation were monitored over a longer time period than the cAMP accumulation assays, so it is possible that the agonists tested were degrading over time leading to loss of response. We have previously demonstrated that for treprostinil and MRE-269 > 90% of the original concentration of agonist remains after 24 h incubation with HLF (unpublished data), suggesting that it is unlikely that ligand degradation is contributing to the effect we have seen.Another aspect of GPCR signalling that may be contributing to the responses we have observed is the ability to signal through multiple G proteins or effector molecules. It has now been well characterized that β-arrestin, once recruited to a GPCR, may initiate a cascade of G protein-independent signalling , the bes2 adrenoceptor agonists, which suggests a role for signalling kinetics in mediating chronic phenotypic events. This has been demonstrated previously for the regulation of gene transcription after GPCR activation, where the kinetics or duration of second messenger responses was shown to drive transcriptional activity [The temporal characteristics of the cAMP response can be controlled directly through the kinetics or duration of the cAMP response, or through time-dependent internalization and desensitization of activated receptors. Agonists such as treprostinil and iloprost which are full agonists in the cAMP accumulation assay may undergo a higher degree of desensitization than the partial agonist MRE-269. This could explain why there is a reversal of intrinsic activity for these ligands in the two assays. However, recent evidence from chronic cAMP studies suggests that regardless of their efficacy, all agonists will produce the same relative degree of functional desensitization . In suppactivity , 32.i-coupled S1P1 receptor [2 adrenoceptor [Compartmentalized cAMP signalling may also contribute to receptor/ligand-dependent downstream effects. Selective activation of specific intracellular pools of cAMP can occur through physical intracellular barriers, such as the A-kinase-anchoring proteins (AKAP) –35, or treceptor  and morenoceptor . Further2, EP2, EP4, and A2B receptors have the potential to inhibit pro-fibrotic processes such as fibroblast proliferation and differentiation, implying a potential for disease modifying effects in IPF. From these results it appears that although the cAMP response is important in driving the anti-fibrotic effects we have observed, there appears to be a threshold of cAMP that is required to inhibit proliferation and differentiation over which any further increases of cAMP do not drive further inhibition. For this reason, the magnitude of acute, global cAMP accumulation does not accurately predict the degree of inhibition of proliferation or differentiation suggesting additional factors, such as the spatio-temporal control of cAMP are important. Further research is required to understand this disconnect, which will aid in the discovery of novel, therapeutically beneficial treatments for IPF.In conclusion, in these studies we have demonstrated that selected agonists at the IP, βAdditional file 1:Additional data describing full GPCR expression analysis in human lung fibroblasts and chemical structures of ligands used in these studies. Further characterization of these ligands in an imaging-based nuclear count assay and cell viability assay is presented. (DOCX 1357 kb)


Idiopathic pulmonary fibrosis (IPF), the most common form of idiopathic interstitial pneumonia (IIP), is characterized by irreversible scarring of the lung parenchyma and progressive decline in lung function leading to eventual respiratory failure. The prognosis of IPF is poor with a median survival of 3–5 years after diagnosis and no curative medical therapies. Although the pathogenesis of IPF is not well understood, there is a growing body of evidence that genetic factors contribute to disease risk. Recent studies have identified common and rare genetic variants associated with both sporadic and familial forms of pulmonary fibrosis, with at least one-third of the risk for developing fibrotic IIP explained by common genetic variants. The IPF-associated genetic loci discovered to date are implicated in diverse biological processes, including alveolar stability, host defense, cell–cell barrier function, and cell senescence. In addition, some common variants have also been associated with distinct clinical phenotypes. Better understanding of how genetic variation plays a role in disease risk and phenotype could identify potential therapeutic targets and inform clinical decision-making. In addition, clinical studies should be designed controlling for the genetic backgrounds of subjects, since clinical outcomes and therapeutic responses may differ by genotype. Further understanding of these differences will allow the development of personalized approaches to the IPF management. Idiopathic pulmonary fibrosis (IPF) is the most common of the idiopathic interstitial pneumonias (IIPs). IPF is characterized by progressive scarring of the lung parenchyma, which leads to dyspnea and declining pulmonary function and eventually to respiratory failure. The median survival after diagnosis of IPF is 3–5 years . In 2011Numerous epidemiologic and genetic studies illustrate that genetic and environmental factors contribute to the risk of IPF , 6. The In this review, we briefly discuss the current literature regarding the role of common and rare variants in disease pathogenesis and prognosis and how this may influence clinical management in the future. Genetic variants and loci associated with IPF involve abnormalities in alveolar stability, host defense, cell–cell barrier function, and cell senescence, all of which are all thought to contribute to the pathogenesis of IPF. We conclude by discussing how treatment decisions might be affected by these findings and how better understanding of genetic variation and disease could allow for a more personalized approach to the treatment of IPF.SFTPC (SFTPA1 (SFTPA2 (ABCA3)  play a role in FIP   , and NAF3) , DSP (6q24), OBFC1 (10q24), ATP11A (13q34), DPP9 (19q13), and chromosomal regions 7q22 and 15q14-15 among others that have been nominally associated  also appear to play a role in FIP risk . The moson study  and has on study , 11. In MUC5B promoter variant rs35705950 is a common variant with a large effect size and therefore accounts for a substantial risk in IPF. In fact, it has been estimated the MUC5B promoter variant accounts for 30% of the risk of developing IPF  have been associated with pulmonary fibrosis  have also been linked to FIP  of alveolar type II cells (AECII) and transported to and stored in the lamellar bodies until secretion into the alveolar space , 26. Rarfibrosis . SP-C isfficking , 26. Codfficking . Mutatiod to FIP  and haved to FIP , 55. Rarfamilies , 56. ABCembranes , 57. In ignaling . These mIn 2011, Lawson et al.  demonstrTERT) and telomerase RNA (encoded by TERC) (TERT and TERC)   , 37. Mutby TERC) . More rend TERC)  and telod RTELI) , 21. Thed RTELI) , 63 in pd RTELI) –34. Thus% of FIP  and in 1adic IPF .TERT or TERC, had short telomeres. In addition, all subjects within this specific study who had a mutation in TERT or TERC and pulmonary fibrosis also had short telomeres  and homozygous (TT) individuals had an odds ratio for developing disease of 6.8 and 20.8 for FIP, and 9.0 and 21.8 for IPF, respectively, supporting the strength of the SNP’s association with development of both IPF and FIP   on chromosome 11 that was associated with both FIP and IPF . The SNP and FIP . MUC5B eal cells , 69. The cohorts , 70–74,  cohorts . Most re cohorts .MUC5B expression in IPF is localized in the distal airway, respiratory bronchiole, honeycomb cyst  studied in the Framingham cohort  region in IPF –89. The DSP and DPP9. DSP gene expression was increased in lung tissue of individuals with IIP and varied by genotype for a variant in intron 5 , may play a role in predicting disease outcomes and have prognostic implications. Short telomeres (<10th percentile adjusted for age) have been identified in a considerable portion of IPF patients, regardless of genetic mutations  and common  in TOLLIP is protective and associated with reduced susceptibility to IPF. However, those who developed IPF despite having the protective allele had increased mortality  . More specifically, of those that received NAC, subjects with TT genotype for rs3750920 (TOLLIP) had decreased risk for the trial’s composite end point of death, hospitalization, or 10% decrement in forced vital capacity. In contrast, subjects with the CC genotype for rs3750920 had increased risk for the composite endpoints of the NAC intervention study  , it is p therapy . Oldham  therapy  found thon study . While Non study , it is pgenotype . More prThis review focuses on the relationship between genetic variants and IPF. In addition to sequence variation, epigenetic changes (such as DNA methylation) –104 and Given the growing body of evidence that genetic variants influence disease risk as well as disease progression and clinically meaningful patient outcomes, it will be critical to account for genetic variation in future clinical trials. Such prospective studies and analyses that focus on the relationship between genotype and therapeutic response will be crucial in personalizing and improving IPF therapy.AK and SM researched and wrote first draft of manuscript; DS edited and revised document. All the authors read and approved final version of manuscript.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.


Regulatory T cells (Tregs) are crucial in maintaining immune tolerance and immune homeostasis, but their role in idiopathic pulmonary fibrosis (IPF) is unclear. This study was designed to explore the role of Tregs in IPF.Percentages of Tregs and their subpopulations in peripheral blood (PB) and bronchoalveolar lavage (BAL) samples were determined by flow cytometry in 29 patients with IPF, 19 patients with primary Sjögren’s syndrome-related interstitial pneumonia (pSS-IP), and 23 healthy controls (HCs).+CD25+Foxp3+ Treg percentages among patients with IPF, pSS-IP, or HCs. However, activated Treg (aTreg) fractions among CD4+ T cells increased significantly in IPF compared with pSS-IP or HCs. Being consistent with the result from the PB, aTreg fractions among CD4+ T cells in IPF also increased significantly compared with pSS-IP or HCs, accompanied by increased fraction III compared with HCs in BAL. IPF patients had lower levels of resting Tregs (rTregs) from the thymus than did HCs, whereas aTreg levels originating from the thymus did not significantly differ from HCs. Both rTregs and aTregs proliferated in IPF, with aTregs being more proliferative than rTregs. Both rTregs and aTregs significantly inhibited proliferation of CD4+ T lymphocytes in vitro. The percentage of aTregs was correlated negatively with predicted diffusing capacity values for carbon monoxide and positively with GAP index in IPF.In peripheral blood, no difference was found in CD4Our study showed the imbalance between subpopulations of Tregs in IPF. Increased aTregs proportion in the peripheral blood correlated inversely with disease severity. Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal chronic lung fibrosis disease of unknown cause with the histopathological features of usual interstitial pneumonia (UIP) . AccumulAlthough it has been accepted that disturbed immune homeostasis plays a significant role in the pathogenesis and/or progression of IPF, the underlying mechanisms remains unclear. IPF has a different fibrotic process than connective tissue disease-related interstitial pneumonia (CTD-IP). It has been proposed that the devastating fibrotic response in IPF is driven by abnormally activated alveolar epithelial cells  rather t1 and platelet-derived growth factor (PDGF)-B [Regulatory T cells (Tregs), a subpopulation of T cells, have immunosuppressive effects that maintain immune tolerance and immune response homeostasis . Accumul(PDGF)-B .+/CD25hi+/Foxp3+ Tregs were retained in the lungs of bleomycin-treated CCR7−/− mice, which was consistent with an ameliorated remodelling response to bleomycin-induced injury in the lungs [Early studies showed that CD4he lungs . The numhe lungs . In conthe lungs , 12.+CD25+Foxp3+ to define Tregs. However, recent research has demonstrated that these classical-defined Tregs are heterogeneous and separable into three functionally and phenotypically distinct subpopulations: CD45RA+/CD25++ resting Tregs (rTregs) and CD45RA−/CD25+++ activated Tregs (aTregs), both of which are suppressive in vitro, and cytokine-secreting CD45RA−/CD25++ T cells (Fr III), which are pro-inflammatory [Most of the studies used CD4ammatory . Based oammatory –16 and iammatory –19. Receammatory , which iWe recruited 29 consecutive Chinese Han patients with newly diagnosed IPF, 19 patients with primary Sjögren’s syndrome-related interstitial pneumonia (pSS-IP), and 23 healthy controls (HCs) at Beijing Chao-Yang Hospital, Capital Medical University, China (Table Diagnosis of IPF was based on the American Thoracic Society (ATS)/European Respiratory Society (ERS)/Japanese Respiratory Society (JRS)/Latin American Thoracic Association (ALAT) statement . PatientPeripheral blood (PB) samples were obtained from each subject and were processed to obtain peripheral blood mononuclear cells (PBMCs) by density centrifugation. BAL was performed and processed as previously described –24 in 7 5 cells were acquired for subsequent data analysis.Fresh PBMCs and BAL cells were stained with the following antibodies: CD45RA-FITC, CD45RA-APC, CD25-PE, CD25-PerCP-Cy5.5, CD4-FITC, CD4-PE-cy7, CD4-APC, CD31-PE, and matched isotypic controls and incubated for 30 min at 4 °C in dark room. For intracellular staining, cells were fixed, permeabilized, and stained with Foxp3-PE and Ki-67-FITC, according to the manufacturer’s instructions. All antibodies were purchased from BD Biosciences or Pharmigen  and e-Bioscience . Data acquisition and analysis were performed with a FACSCalibur, which was equipped with CellQuest Pro software . Approximate 10+ T cells were firstly isolated by positive selection of PBMCs (obtained from 30 ml whole blood) labeled with magnetic-bead conjugated anti-human CD25 mAbs using MACS MultiSort Kit according to manufacturer’s instructions (Miltenyi) from IPF patients. Purified CD25+ T cells were stained with CD4, CD25, and CD45RA antibodies, and then sorted into CD4+CD25++CD45RA+ cells , CD4+CD25+++CD45RA− cells , and CD4+CD25++CD45RA− cells  using a FACS Aria II flow cytometer (Becton Dickinson). The purity of the Treg subsets was more than 95%. The CD4+CD25− cells  from healthy donors (10 ml whole blood) can also be isolated using magnetic-beads conjugated anti-human CD25 mAbs and anti-human CD4 mAbs.For functional assays, CD25+CD25− responder cells (2 × 104) from healthy donors were labeled with 1 μM CFSE  and were then cocultured with (1 × 104) unlabeled, sorted rTreg, aTreg and Fr III cells at a 1:2 Treg subpopulations/CD4+CD25− responder cell ratio in anti-CD3  coated plates in the presence of soluble anti-CD28  for 72 h at 37 °C and 5% CO2 in complete medium . CFSE-labeled cells was assessed by flow cytometry.CD4The multidimensional GAP index is a simple and reliable tool for disease severity stratification in IPF. We calculated the GAP score for every IPF patient according to the method reported by Ley et al. .t-test, Wilcoxon rank-sum test, or a chi-square test as appropriate. Correlations were assessed using a Pearson correlation test or Spearman’s rank test. A p value <0.05 was considered significant.All analyses were performed with SPSS for Windows V16.0 . Values are presented as the mean ± standard (SD) or as the median and IQR when appropriate. Groups were compared using analysis of variance, Student’s p < 0.001). Pulmonary function values did not significantly differ between the IPF and pSS-IP groups.The characteristics of 29 patients with IPF, 19 with pSS-IP and 23 HCs are summarized in Table +CD25++ in different groups. Compared with HCs, patients with IPF or pSS-IP did not have significant differences in the percentages of CD4+CD25++ cells , aTregs  and the cytokine-secreting subpopulation , as described previously . Strikingly, compared with patients with pSS-IP and HCs, patients with IPF had an increase in the aTreg fraction  were observed in aTregs from IPF patients and HCs  and DLCO that are used to predict IPF severity. As shown in Fig. −   and HCs   Fig. . More im02) Fig. .Fig. 4AnIPF is a chronic, progressive fibroproliferative interstitial pneumonia of unknown aetiology. Various studies have clarified the crucial roles of Tregs in many diseases, but contradictory results have been obtained from both murine models and IPF patients. Here, by using the new definition of Treg subpopulations, we showed that, compared with HCs and the patients with pSS-IP, patients with IPF had larger fractions of circulating aTregs, which was negatively correlated with DLCO predicted values and positively correlated with GAP index.+/CD25+ Treg cells was observed in patients with pSS. Here, we also observed a significant decrease in the percentages of rTregs in patients with pSS-IP, while the percentages of aTregs remained comparable with the HCs. This finding indicated that inflammatory responses are related to a loss of rTregs in these patients. Similarly, IPF patients also had decreased percentages of rTregs. However, a dramatic increase in the percentages of aTregs was noticed. More importantly, a more dramatic increase in aTreg frequencies was observed in BAL samples of the patients with IPF. Thus, the disturbed homeostasis patterns between the IPF and pSS-IP patients were quite different. An increase in aTregs is a hallmark of disturbed immune homeostasis in IPF patients and might compensate for the loss of the rTreg fraction.One of the most important findings of the present study is exploring a characterized disturbance of Treg subpopulations in patients with IPF. Consistent with the protective effects in pSS and other autoimmune diseases –31, a sl+/CD25+ Treg cells [More importantly, although IPF patients display distinct defects in Treg subpopulations compared to patients with pSS-IP, a negative correlation was also seen between circulating aTregs and DLCO in IPF patients. Decreased rTregs and increased aTregs both showed detrimental effects in patients with pSS-IP and with IPF, respectively, which indicated that varying mechanisms contribute to fibrosis formation in these two diseases. In addition to a decreased number of Treg cells in pSS patients, in vitro functional assays showed decreased suppressive activity of CD4eg cells . Such deBy contrast, in addition to an increase in aTreg cells, our in vitro functional analysis demonstrates that rTregs from IPF patients are functionally immunosuppressive. Our study showed that in addition to increased percentages of aTregs, inflammatory BAL Fr III cells were also increased. Despite the elevation of these two subpopulations, the aTreg/Fr III ratio in IPF patients was higher than in pSS-IP patients but comparable to that in HCs. From the above data, immune homeostasis seems to remain intact in IPF patients, although inflammatory and anti-inflammatory mechanisms were activated. This might explain why IPF patients are unresponsive to corticosteroids and/or immunosuppressive agents, whereas pSS-IP patients might benefit from them.+ rTregs in IPF patients was significantly lower than in healthy controls, whereas CD31+ aTregs levels did not significantly differ between IPF patients and healthy controls. These results suggest an impaired thymic output of Tregs in IPF patients. The extensive turnover of the existing aTreg cells or by peripheral conversion of rTreg cells or non-Treg cells into aTreg cells have been proved to maintain the aTregs pool. The percentages of the CD31+ fraction in aTregs were low and in comparable level in IPF patients and HCs, indicating IPF might not affect conversion of rTregs to aTregs and contribute partly to the maintenance of aTreg cells pool. Actually, rTregs and aTregs represent essentially distinct populations from the genomic standpoint [+ fractions in aTregs and rTregs in IPF patients compared with healthy controls. Thus, hyper-proliferation at least partly contributed to the high levels of circulating aTregs in IPF patients. These observations hint that a shift in the homeostatic composition of Treg subsets related to an impaired thymic-dependent de novo generation of recent thymic emigrant rTreg cells with a compensatory expansion of aTreg cells may contribute to imbalance of Treg subsets in IPF patients. What is noteworthy is that aTregs represent a highly differentiated population, characterized by short telomeres, inability to upregulate telomerase and susceptibility to apoptosis [−/CD4+ non-Tregs [The disturbed homeostasis among Treg subpopulations in IPF may reflect cell derivation. All T cells, including these subpopulations, are derived from progenitors in the bone marrow and differentiate in the thymus . Under vandpoint .In presepoptosis . Therefoon-Tregs  or from on-Tregs . It is o1 [+ T cells in IPF patients was inversely correlated with DLCO predicted values. The multidimensional GAP  index and staging system is a simple method for informing prognosis, helping guide management decisions in patients with IPF [Of note, it has been shown that Tregs can contribute to lung fibrosis by stimulating fibroblasts through the secretion of PDGF-B under non-inflammatory conditions and regulate detrimental T-cell activity during inflammation-related fibrosis , 6. More1 . Thus, iwith IPF . FurtherOur study has some limitations that deserve comment. One limitation of this study is that the potential role of aTreg cells in chronic fibrotic lungs was not fully declared. Further research work is warranted to disclose whether the cells are per se that induce fibrogenesis or it is the pro-fibrotic microenvironment that shifts them towards a pro-fibrotic phenotype. Secondly, despite the appreciable effort of obtaining BAL samples, the results from the small size of the patients obtaining BAL may be compromised.In conclusion, our study provided further evidence for the role of adaptive immunity in the pathogenesis of IPF and showed an imbalance among subpopulations of Tregs in IPF. Importantly, the increased aTregs in the PB correlate inversely with disease severity. Our study may suggest Tregs as potential future therapeutic targets by restoring their homeostasis. Treg subpopulations may be promising prognostic factors for IPF as well.


Emission of the trace gas dimethylsulphide (DMS) from the ocean influences the chemical and optical properties of the atmosphere, and the olfactory landscape for foraging marine birds, turtles and mammals. DMS concentration has been seen to vary across seasons and latitudes with plankton taxonomy and activity, and following the seascape of ocean’s physics. However, whether and how does it vary at the time scales of meteorology and day-night cycles is largely unknown. Here we used high-resolution measurements over time and depth within coherent water patches in the open sea to show that DMS concentration responded rapidly but resiliently to mesoscale meteorological perturbation. Further, it varied over diel cycles in conjunction with rhythmic photobiological indicators in phytoplankton. Combining data and modelling, we show that sunlight switches and tunes the balance between net biological production and abiotic losses. This is an outstanding example of how biological diel rhythms affect biogeochemical processes. The knowledge gained about the trace gas dimethylsulphide (DMS) and its main biological precursor, the algal osmolyte dimethylsulphoniopropionate (DMSP), over the last 40 years makes these compounds some of the best-studied organic substances in the world’s oceans139101012314Oceanic DMS concentration depends on the interplay of many processes: DMS is produced mainly by enzymatic degradation of the phytoplankton osmolyte DMSP with involvement of the entire planktonic food web16241524a (chla) fluorescence but traditional analytical methods for DMS take several minutes for a single measurement. This study takes advantage of high frequency mass spectrometric and optical measurements, coupled to continuous vertical sampling with a recently developed profiler to allow a view of upper ocean DMS cycling at unprecedented resolution. Here we report results from two Lagrangian studies conducted in open oligotrophic waters of the western Mediterranean Sea illustrating the complex response of DMS cycling to changes in meteorology, solar radiation, and phytoplankton photophysiology.Few studies have addressed the short-term variability of DMS on time scales of hours to weeks across phases of natural and fertilized phytoplankton blooms262728293132−1), with diurnal variations due to basin-scale sea breeze circulation. There were two episodes of increased winds. On 14 September winds increased to 7–8 m s−1 for almost 12 hours and on 18–19 September elevated winds associated with a storm exceeded 14 m s−1 and obliged to stop the cruise for 30 hours. Calm winds and clear skies followed the storm for the last 2.5 days of the cruise  increased gradually on calm sunny days, and decreased by about 70% during the stormy period on 18–19 September  and DMS in response to meteorological perturbation, vertical mixing, and re-stratification.Surface DMS concentration time series also exhibited a strong diurnal cycle. DMS levels increased during the day and decreased at night, with a minimum near dawn and maximum around dusk . Mean sueptember . On the −1 and a maximum of 7.6 m s−1 (−1) were considerably higher than in September, in agreement with the typical seasonality in the regionDuring a brief, 2-day cruise in late May, wind speed was lower than during the September cruise, with a minimum of 0.27 m s.6 m s−1 . DMS conSynechococcus and prymnesiophytes. Phytoplankton at the deep chla maximum (55 m approx.) were dominated by Prochlorococcus, dinoflagellates and prymnesiophytes. In May, the surface assemblage was rather similar to that in September, but the deep chla maximum assemblage was dominated by prymnesiophytes, dinoflagellates, Synechococcus and pelagophytes. Surface chla concentrations were similar during the two cruises (nearly 0.1 μg L−1), but the concentration at the deep chla maximum was 1.5–2 times as high in May , within the water layer that in oligotrophic waters typically exhibits the maximum primary production rates. Chla concentrations showed a clear deep maximum around 50–60 m, and increased over daytime through most of the water column. In May, a slight chla concentration increase from dawn to dusk was only noticeable at the deep maximum (50 m). DMSPt behaved similarly at depth but the concentration slightly decreased at surface during daytime. Mixed-layer DMS also decreased during daytime.Vertical profiles reveal a more complete picture of the processes controlling surface DMS variability. a peaks at depth , DMSPt peaks from right above the chla peak to right below the mixed layer, and DMS peaks up in the mixed layer, or right below.The profiles described above portray the typical open ocean vertical distributions in oligotrophic watersin vivo fluorescence measured with an FRRf (as a proxy for chla) were recorded  and increased in the afternoon towards dusk. Days after the windstorm, DMS had recovered its diurnal cycle within the upper 30 m.The use of continuous data recording in continuous profiling mode permitted monitoring the vertical changes of target variables at much higher temporal resolution than CTD-rosette sampling profiles. DMS and recorded . Unfortu maximum  could ba maximum . After ta-rich layer exhibited oscillations, likely due to internal waves, but no clear diel pattern. By contrast, at the surface fluorescence was higher during the night and lower during the day, a phenomenon that might be indicative of phytoplankton photoacclimation cyclesIn May, vertical turbulent mixing was deeper . The latter displayed two features typically observed in stratified surface waters: 1) a midday depression, caused by high irradiance and the subsequent increase in non-photochemical quenching and possible photosystem damage, and 2) pre-dawn and pre-dusk peaks, related to changes in the photosystem electron acceptor pools. DMSPt and the beam attenuation coefficient due to particles at 660 nm (cp), also displayed diel periodicity. A regular cp cycle with dawn minima and pre-dusk maxima was found in all three intensive sampling periods. This feature is ubiquitous in the open ocean and stems mainly from the increase in cell size of phytoplankton due to photosynthesis35p is most responsive to changes in the particle population of sizes ranging 0.5–20 μm, and especially around 5 μmp decrease was probably related to synchronized cell division at nightp but was lagged in time and less repeatable: after an early morning plateau or decrease, DMSPt increased steadily until dusk or into the evening, and decreased at night. Thus, the trend reversal points of the diel DMS pattern  coincided with switching points marking the diel phasing of phytoplankton physiology and cell growth, and to a lesser extent to changes in the sign of net DMSPt variations.The fact that the diel variation series of surface DMS concentrations had dawn and dusk as the minima/maxima suggests the involvement of photobiological processes with diel rhythms. The General Ocean Turbulence Model (GOTM) was used to compute vertical fields of the turbulent eddy diffusivity over time for the three intensive studies . The mod−2 h−1) and on 23 May early morning (−2.3 μmol m−2 h−1). Upward DMS transport events were observed on 22 September midday (2.6 μmol m−2 h−1) and 24 May midnight (2.5 μmol m−2 h−1). The average transport fluxes across the base of the mixing layer (defined with the Δσt = 0.05 kg m−3 criterion) were smaller than ±0.07 μmol m−2 h−1in the three intensive studies, which represented contributions to the volumetric DMS budget in the mixing layer smaller than ±0.008 nM h−1. Calculated DMS ventilation fluxes ranged up to 0.27 μmol m−2 h−1 in September and up to 0.5 μmol m−2 h−1 in May  occurred mainly during short episodes (2–4 h long) associated to nighttime convection and/or wind outbursts. Downward DMS transport events occurred on 14 and 21 September, early morning  during the mid-day, and negative values (net consumption) in the night. In May, NPBIO rates ranged from −0.03 to 0.7 nmol L−1 h−1 and were again at its highest at the surface (<5 m) during the day. Vertical gradients in NPBIO were less clear in May.The turbulent diffusivity fields and the abiotic DMS loss rates were used to compute net biological DMS production  at high vertical-temporal resolution by budgeting . In Septin situ (overboard) for 6–8 h during the September cruise. The results show remarkable agreement in the magnitude of daytime NPBIO  or incubated me NPBIO  and the me NPBIO .The results of this study reveal the strong effects of mesoscale meteorological forcing on DMS production and concentration in the surface sea. They also illustrate why diurnal cycling and finely resolved vertical distributions, both resulting from water mixing dynamics and sunlight-synchronized microbial activities, must be taken into account when intending to predict ocean emissions of DMS.A notable difference between the two cruises was that in September the maximum DMS concentration was near the surface, whereas in May there was a subsurface maximum below the mixing layer depth. Thermocline-associated DMS maxima are quite common in the open ocean1831333839In September, the passage of a windstorm and subsequent return to the previous calm conditions provided the opportunity to monitor the effects of the perturbation and the resilience of the DMS-producing ecosystem. During the days prior to the storm, prolonged high insolation under weak wind caused gradual DMS accumulation. Then, the persistent wind reduced the surface DMS concentration to one third, mainly through ventilation to the atmosphere but also by turbulent mixing to deeper water. The overall loss of the DMS burden integrated down to 30 m depth (the deepest extent of wind-driven mixing according to temperature profiles) was in the order of 30%. Noteworthy, a similar coupling of DMS variability to synoptic-scale meteorological forcing had been observed in an early summer coccolithophore bloom in the North AtlanticThe issue of mixed layer dynamics is relevant to understand DMS dynamics in the global oceans both at present and in response to global warming. One might think that mid and long-term changes in wind speed are first order drivers of trace gas emissions because gas transfer velocities are strongly dependent on wind speed. However, the magnitude of emission fluxes depends on trace gas concentrations as well, and persistent wind depletes concentration from the ocean by ventilation; besides, windy regions generally have deeply mixed waters where trace gases like DMS can hardly build up as they do in stratified waters−1 d−1 equals DMS concentration in nmol L−1), then microbial DMS consumption (BC) can be estimated roughly as BC = GP - NPBIO and compared with the other loss rates. In September before the storm, BC accounted for some 37% of the daily DMS loss, PHOTO represented 38% of the loss and VENT and transport were 16% and 9%, respectively. After the storm, these figures were 36%, 49% and 15%, and average transport resulted in DMS injection into the UML. In May, loss shares were 50% (BC), 40% (PHOTO), 9% (VENT) and 1% (TRANSPORT). In these shallow MLs, therefore, microbial consumption and photolysis competed as the main loss processes, well above export into air above or water below. Photolysis rates equal or higher than microbial consumption rates had already been reported in the North Atlantic, also coinciding with shallow ML and high irradianceWith the detailed data obtained by observations and modelling in the intensive studies, we can attempt the 48-h average budgets of the process rates that control DMS concentrations in the UML . BiologiSunlight exerts a strong influence on the distribution and biogeochemical cycling of DMS1624174243444633PSII, coinciding with indicators of photoacclimation.In summary, the high-resolution field results reported here align with the aforementioned previous observations of the strong influence of solar radiation onto the DMS cycle, and reveal for the first time that this influence operates in the short term, sub-daily to weekly. First, surface DMS concentration increased gradually in sunny calm days, even after a strong perturbation. Second, DMS concentrations peaked upper in the water column than those of DMSP and chlThis study illustrates the value of detailed automated profiling in a Lagrangian sampling framework in terms of revealing the dynamics controlling DMS concentrations and fluxes and the intimate links between plankton photophysiological clocks and the temporal cycles of DMS. The results highlight the causes of spatial/temporal variability54−1.The two cruises were conducted aboard R/V García del Cid in the Western Mediterranean Sea, in September 2011 and May 2012. Three Lagrangian drifters were deployed to track the movement of the upper 15-m water layer. Each drifter consisted of a spherical floatable enclosure that contained a GPS and an emitter, from which 10 m cylindrical drogues hanged 5 m below the sphere. We decided to track the upper 15 m water layer because, on the basis of previous cruises, we expected the mixing layer depth to be between 5 and 20 m. The drifters sent their position every 30 minutes, and all ship operations were conducted next to them. Both cruises were held in the same region, ca. 45 nautical miles from the coast, within the core of a cyclonic eddy over a water-column depth of ca. 2000 m . SeawateSolar radiation and wind speed, among other variables, were measured with an Aanderaa Instruments automated meteorological station on board. A Solar Radiation Sensor 2770 provided global radiation in the wavelength range 0.3–2500 μm. Wind speed and wind direction were measured with a three-cup Sensor 2740/2740EX and a wind vane 3590/3590EX, respectively.−1, and precision was around 10%. Total (particulate and dissolved) DMSP concentration was measured in 3 mL discrete samples by gas chromatography (GC) coupled to flame photometric detection (FPD)a concentrations were determined by filtration of 150 mL of seawater through GF/F, extraction in acetone  and measurement in a Turner Designs fluorometer. Taxonomic and photoprotective pigments were analysed by HPLC  after GF/F filtration of 1–2 L of seawater and extraction with methanola fluorescence data  were derived from the fluorescence induction curvePSII =  (F′m − Fs)/F′m, where Fs is the steady state in vivo Chla fluorescence of phytoplankton and F´m is the maximum yield of fluorescence during the illumination. This correlated with variations in the quantum yield of photosynthesis. Every day around noon, a PRR-800 multichannel profiling radiometer  was deployed overboard to profile underwater irradiances in the PAR, UVA and UVB bands. Extinction or diffuse attenuation coefficients of downward irradiance (Kd) were calculated as the linear regression between ln-transformed spectral irradiance and depth (z) in the optically homogeneous surface layer. All relevant optical data are described in detail elsewheret) was calculated using high resolution CTD profile data. This was used to compute vertical profiles of the Brunt-Väisälä buoyancy frequency (s−1). High-resolution profiles were used to compute the mixing and mixed layer depths, defined by a given density difference (Δσt) from the reference depth (2 m). A Δσt of 0.05 kg m−3 best matched the base of the high-turbulence mixing layer  was used for continuous DMS measurements from surface waters and across the vertical column during intensive studies . The insng layer ; a Δσt orequency .in situ temperature. One layer of a neutral mesh (60% transmittance) was used to simulate the average irradiance of the upper mixed layer. DMS concentration was measured at the beginning and end of the incubation. Changes in DMS concentration, once corrected for photochemical loss, corresponded to net biological production ratein situ light fields at different depths and NPBIO was calculated similarly  was used to compute vertical profiles of turbulent diffusivity during the 3 intensive experiments  spatial resolution and 1 min temporal resolution. They were subsequently smoothed using the running average method.The General Ocean Turbulence Model  and depth (z), DMS concentration is the net result of production and consumption processes:At any time  would be of the order of 0.01. This in turn implies that vertical transport by diffusivity is 2 orders of magnitude larger than by non-turbulent advection. Based on this calculation it would seem justified that we chose to neglect non-turbulent transport.Vertical transport by non-turbulent advection is considered negligible. Since our model is 1D, we do not have any information on the 3D velocity structure of the eddy . Some A horizontal transport term is not included because the same water patch was sampled in Lagrangian mode over the entire duration of the cruises, and therefore horizontal advection was set to zero by definition. Besides, horizontal diffusivity was assumed negligible because strong horizontal gradients were not expected. We did not map the region for DMS concentration, so, we cannot prove this assumption to be true, but support is to be found in ref. Ventilation applies only to the very upper water layer, from where its effects are “redistributed” by turbulent diffusion. GP and BC can be merged into a single term called Net Biological DMS production (NPBIO):d,o,t) and the underwater light extinction coefficients (Kd) were combined to obtain the amount of radiation available at each t and z :For the calculation of DMS photolysis (PHOTO), total surface irradiance  was used for downward photolysis propagation because the spectral peak of DMS photolysis in the UML has its maximum yield at this wavelengthw,DMS, cm h−1):w,DMS was computed using wind speed data from the ship’s meteo station, SST and surface DMS concentrations, following ref. K10 = wind speed at 10 m (m s−1) and Sc = Schmidt number of DMS, calculated from SSTz) diagnosed with the GOTM and vertical DMS gradient as:The final depth-time matrix of NPBIO was then obtained applying the following equation at a resolution of 1 m  and 1 min:We assume that the main sources of uncertainty are DMS measurements themselves and photolysis, the loss term that drives the budgets. This justifies not taking into account uncertainties derived from sea-air flux and vertical mixing parameterizations. Uncertainty in 2-m and 30-min bin DMS concentration averages, as calculated by ref. We assume no uncertainty in the sea-air flux parameterization. Note however that different parameterizations may differ by>50%.The CV of maximal subsurface photolysis rate constants (CV_Kmax) is 9% (SUMMER1) and 19% (SUMMER2). We can assume that uncertainties in irradiance cancel each other, since photolysis is vertically propagated following the quotient Ed,z,t/Ed,0,max. Thus, the uncertainty associated with the photolysis rate is calculated by adding the CVs in quadrature:CV_DMS of 11% becomes 15.5% when computing the DMS gradient (dDMS = DMS_z1-DMS_z2), since errors add in quadrature.We assume that the error in Kz is the Kz range through a 2 m interval encompassing the ML base, which is the depth horizon used to compute DMS transport. CV_Kz is 48%, 88% and 86%, respectively, for pre-storm, post-storm and May intensive studies.Error is propagated by adding in quadrature the error of loss terms plus the error in dDMS, the DMS change between two time steps, which is 15.5%.How to cite this article: Royer, S.-J. et al. A high-resolution time-depth view of dimethylsulphide cycling in the surface sea. Sci. Rep.6, 32325; doi: 10.1038/srep32325 (2016).


Increased β cell PD-L1 expression was dependent on T cell infiltration, as β cells from Rag1-deficient mice lacked PD-L1. Using Rag1-deficient NOD mouse islets, we determined that IFN-γ promotes β cell PD-L1 expression. We performed analogous experiments using human samples, and found a significant increase in β cell PD-L1 expression in type 1 diabetic samples compared to type 2 diabetic, autoantibody positive, and non-diabetic samples. Among type 1 diabetic samples, β cell PD-L1 expression correlated with insulitis. In vitro experiments with human islets from non-diabetic individuals showed that IFN-γ promoted β cell PD-L1 expression. These results suggest that insulin-producing β cells respond to pancreatic inflammation and IFN-γ production by upregulating PD-L1 expression to limit self-reactive T cells.Type 1 diabetes is caused by autoreactive T cell-mediated β cell destruction. Even though co-inhibitory receptor programmed death-1 (PD-1) restrains autoimmunity, the expression and regulation of its cognate ligands on β cell remains unknown. Here, we interrogated β cell-intrinsic programmed death ligand-1 (PD-L1) expression in mouse and human islets. We measured a significant increase in the level of PD-L1 surface expression and the frequency of PD-L1 Genetic deficiency of PDCD1 in C57BL/6 and BALB/c mice leads to spontaneous lupus-like disease or autoimmune cardiomyopathy, respectively7, while non-obese diabetic (NOD) mice lacking either PD-1 or PD-L1 developed accelerated type 1 diabetes (T1D)6. Antibody blockade experiments suggest that PD-1:PD-L1 interactions, but not PD-1:PD-L2, are necessary for the maintenance of tolerance in the NOD model of T1D14. Several lines of evidence also suggest that the PD-1:PD-L1 pathway plays a role in maintaining islet tolerance in humans as recent onset patients with T1D have elevated gene expression levels of CD274 (PD-L1) in whole-blood RNA analysis15. Additionally, single nucleotide polymorphisms in the PDCD1 or CD274 genes have been associated with T1D18. Finally, adverse events such as rapid autoimmunity including T1D can develop following checkpoint blockade in cancer patients20, further suggesting a role for this inhibitory pathway in autoimmunity.The inhibitory receptor Programmed Death-1 (PD-1) and its ligands Programmed Death Ligand (PD-L) 1 and 2 are critical regulators of immune cell function and autoimmunity21. Many cells throughout the body can express PD-L1 including both hematopoietic and non-hematopoietic cells22. PD-L1 is constitutively expressed on resting T cells, B cells, dendritic cells, and macrophages, and is further upregulated upon cellular activation or in response to cytokines25. Previous work suggests that PD-1:PD-L1 interactions within the pancreas may limit autoimmune diabetes26. Despite this body of knowledge, the timing, location, and specific cellular interactions that are regulated by PD-1:PD-L1 in T1D remain unclear. While previous reports have shown intra-islet PD-L1 expression on infiltrating mononuclear cells27, and suggest a role for non-hematopoietic PD-L1 expression to limit diabetes, it is unclear if β cells themselves express PD-L1 and how this expression is regulated during diabetes progression. Additionally, enforcing PD-L1 expression on β cells under the insulin promoter has shown conflicting results, as NOD mice were protected from disease28 while diabetes-resistant mice were rendered susceptible with insulin promoter-driven PD-L1 expression29.PD-1 is rapidly expressed on the surface of T cells following activation, to diminish their proliferation and effector function upon ligand bindingCD274 promoter contains two interferon regulatory factor-1 (IRF-1) binding sites, and previous work has shown that type 1 and type 2 interferons (IFN) induce PD-L1 expression on T cells, B cells, endothelial cells, epithelial cells, and tumor cells22. We found that IFN-γ and to a lesser extent, IFN-α, promoted increased frequency of PD-L1+ β cells, and increased expression on a per cell basis. Similar to our findings in mice, within human pancreas we found that increased PD-L1 expression correlated with increased inflammatory T cell infiltration in pancreatic lesions. Interestingly, we observed a minor increase in PD-L1 staining in autoantibody positive patients in the absence of overt autoimmune diabetes and found that Th1-associated cytokine IFN-γ modulated PD-L1 expression in vitro on isolated human islets. Taken together, this work illustrates that both mouse and human islet β cells express PD-L1 in response to the same inflammatory cues, which may help delay islet destruction, but is ultimately insufficient to prevent β cell death.In this study, we measured islet β cell PD-L1 expression and regulation during diabetes pathogenesis. The goals of this study were to improve upon previous strategies for flow cytometric analysis of individual, insulin-positive, live β cells, and determine the specific regulators, location, and timing of PD-L1 expression in both mouse and human β cells. We utilized multicolor flow cytometry and epifluorescent microscopy to measure PD-L1 expression on islet β cells during spontaneous diabetes in NOD mice, and found that PD-L1 expression increased as mice approach diabetes onset, and was associated with islet infiltration. We also investigated the effect of cytokines on PD-L1 expression. The −, CD11c−, CD11b−, B220−, F4/80−), live cells, that were positive for intracellular insulin  expression on islet β cells from the pancreas of NOD mice during type 1 diabetes. In NOD mice, β cells were identified as side and forward scatter high, CD45.1 negative, CD4 negative, lineage marker negative . These included sections from patients with type 1 diabetes, autoantibody positive individuals, patients with type 2 diabetes, and non-diabetic controls. Evaluation of these samples by epifluorescent microscopy allowed us to simultaneously evaluate insulitis, defined by the intra-islet presence of one or more CD4+ or CD8+ T cells, and determine the intensity of PD-L1 expression on islet β cells defined by insulin positive cells38 , intracellular insulin-positive cells. Similar to our results using murine islets, Fig. + β cells . We extended our analysis to human islets from non-diabetic, type 2 diabetic, autoantibody positive, and type 1 diabetic human patients and determined that β cells from type 1 diabetic and autoantibody positive patient pancreas sections also have significant PD-L1 associated with T cell infiltration.Previous work has demonstrated PD-L1 expression within the islets39. This intimate regulation may also limit effective T cell inhibition globally throughout the pancreas and account for continued β cell death and eventual loss of islets. It is also true that PD-L1 expression is not uniquely expressed on islet β cells within the pancreas. Results in Fig. 41. Our work suggests that β cell-intrinsic PD-L1 expression is strongly associated with insulitis and driven by IFN-γ and to a lesser extent IFN-α, and likely has a functional significance during disease progression. It is possible that other cytokines involved in T1D but not tested herein also drive PD-L1 expression. For instance, IL-17 has been shown to induce PD-L1 on human colon and prostate cancer cell lines in an NF-κB-dependent manner42. Whether IL-17 has the same effect on β cells remains to be tested.The histological data (both mouse and human) suggests that islet β cell expression of PD-L1 is highly dependent on T cell infiltration. We demonstrate that inflamed islets have the highest level of PD-L1 expression and neighboring islets without significant T cell infiltration do not have strong PD-L1 expression. This result suggests that the cytokines driving PD-L1 act locally and not through a diffuse network, reminiscent of observations in human malignancies where PD-L1 expression is often detected on tumor cells adjacent to immune cell infiltrates43. PD-1 is highly expressed on tumor infiltrating T cells and checkpoint therapy in patients with PD-L1+ tumors has been shown to be effective in early clinical trials43. However, adverse events such as rapid autoimmunity including T1D can develop following checkpoint blockade, further suggesting a role for this inhibitory pathway in regulating autoimmunity19. Therefore, given the current information and PD-L1 expression within the islets, and our previous work showing that nearly all islet antigen-specific CD4+ T cells express PD-1 in the pancreas44, cautious application of PD-1:PD-L1 blockade is warranted. Targeting tumor-specific T cells with checkpoint therapy while restraining autoimmunity is of great importance. Our work suggests that regulation of inflammatory cytokines may be a new avenue of focus to enhance or diminish PD-L1 mediated T cell inhibition, especially when considering checkpoint therapy in individuals with HLA types associated with higher risk of developing autoimmunity or individuals that test positive for islet specific autoantibodies. In one of the three human patients with increased autoantibodies we found a significant increase in PD-L1 expression over control and T2D patients . NOD mice were purchased from Taconic . Diabetes was defined as 2 consecutive blood glucose readings >250 mg/dL. All mice were housed in specific-pathogen free barrier facilities and all animal experiments and methods were performed in accordance with the relevant guidelines and regulations approved by the Institutional Animal Care and Use Committee at the University of Minnesota.NOD.Rag149. To identify PD-L1 expression on β cells, we used the following antibodies: goat anti-mouse PD-L1  at 1:500, AF647 bovine anti-goat  at 1:500, guinea pig anti-insulin  at 1:800, anti-guinea pig IgG (H + L) Cy3  at 1:500, and anti-CD4 BV421 GK1.5  at 1:100. To determine the extent of insulitis, we stained sections with guinea pig anti-insulin (Dako) at 1:1000, anti-guinea pig IgG (H + L) Cy3 or Cy5  at 1:1000, anti-CD4 AF488 GK1.5 at 1:100 and anti-CD8 APC or PE 53–6.7  at 1:100. Slides were mounted with Prolong Gold anti-fade with or without DAPI (Thermo Fisher Scientific). Images were acquired on a Leica DM6000B epifluorescent microscope. Islets were scored by a blinded investigator using the following scale: 0 – no insulitis, 1 – peri-insulitis, 2 – less than 25% of islet mass infiltrated, 3 – less than 75% of islet mass infiltrated, 4 – more than 75% of islet mass infiltrated30.Pancreata were harvested and prepared as describedOCT-embedded fresh frozen human pancreas samples were provided from the Network for Pancreatic Organ Donors with Diabetes (nPOD). De-identified patient information and demographics is listed in Supplemental Table −/− female mice between 7–13 weeks of age, or 5–23 week old NOD mice, as previously described50. Briefly, 3 ml of ice-cold CIzyme  was prepared following manufacturer’s instructions and injected into the common bile duct to inflate the donor pancreata. Pancreata were digested at 37 °C for 17 minutes. Following digestion, islets were washed with HBSS, centrifuged at room temperature at 2000 rpm for 20 min in Lympholyte1.1 , washed with 10% fetal calf serum (FCS)  in HBSS, and hand-counted. Islets were cultured in CMRL 1066 (Thermo Fisher Scientific) containing 1% Glutamax (Thermo Fisher Scientific), 10% FCS and 1% Pen/Strep (Thermo Fisher Scientific). To determine β cell response to pro-inflammatory cytokines, we supplemented cultures of 100 hand-counted pancreatic islets with murine IFN-γ [50 ng/ml], TNF-α [10 ng/ml], IL-1β [17.5 ng/ml], CXCL10 [20 ng/ml], IL-10 [20 ng/ml], TGF-β [10 ng/ml], IFN-α [10 ng/ml], IL-4 [10 ng/ml], IL-12 [20 ng/ml], and IL-21 [20 ng/ml] in non-tissue culture treated 12-well plates as indicated. All cytokines were purchased from Biotechne .Islets were harvested from NOD.Rag152. Human islets were hand-counted and 1000 islets/well were cultured in vitro in CMRL 1066 containing 1% Glutamax, 1% Pen/Strep, 10% pooled human serum with or without human IFN-γ [50 ng/ml] (Biotechne) as indicated. Following overnight incubation at 37 °C containing 5% CO2, islets were dissociated using an 18-gauge needle on 1 ml syringes (BD Biosciences) in non-enzymatic cell dissociation solution . Cells were incubated with Human BD Fc block (# 564219 BD Biosciences) diluted in FACS buffer (2% FCS in PBS) prior to staining.In compliance with federal regulations, islet isolations were performed at the University of Arizona or the University of Louisville utilizing pancreata from deceased donors with research consent. The islet isolation and culture procedures were performed as previously described53. Following fixation and permeabilization (Tonbo Biosciences), cells were stained with guinea pig anti-insulin  at 1:500, then washed and stained with secondary anti-guinea pig IgG (H + L) Cy3 at 1:200.Single-cell suspensions of mouse pancreatic islets were blocked with 2.4G2 in FACS buffer and stained with: anti-CD4 BV650 , anti-CD45.1 APC , anti-CD11b APC-efluor780 (M1/70), anti-B220 APC-efluor780 (RA3-6B2), anti-F4/80 APC-efluor780 (BM8), anti-CD8a APC-efluor780 (53–6.7), anti-CD11c PE-Cy7 (N418), anti-PD-L1 PerCP-efluor710 (MIH5) purchased from Thermo Fisher Scientific, and live/dead BV510 Single-cell suspensions of human pancreatic islets were stained with 5 µl per test of the following antibodies: anti-CD45.RA BV510 (HI100), anti-CD3 PE-Cy7 (SK7) purchased from Biolegend, live/dead ACP-efluor780, anti-CD11b PE-Cy7 (ICRF44), anti-CD20 PE-Cy7 (2H7) purchased from Tonbo Biosciences, anti-CD45RO BV650 (UCHL1), anti-CD11c BUV395 (B-Ly6) from BD Biosciences, anti-PDL1 PerCP-efluor710 (MIH1) purchased from Thermo Fisher Scientific. Following fixation and permeabilization (Tonbo Biosciences), cells were stained with anti-insulin  at 1:500, then washed and stained with secondary anti-guinea pig AF647 at 1:200 (Jackson ImmunoResearch). Data were acquired on FACS Fortessa X-20 flow cytometer (BD Biosciences) and analyzed with FlowJo version 10 .Data were analyzed using Prism 7 . P-values shown for individual figures represent results of two-tailed Student’s T-test or One-Way ANOVA with Tukey post hoc correction between cytokine treatment groups, or between different age groups for percent PD-L1-expressing β cells and PD-L1 geometric mean fluorescent intensity.The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Supplemental Table


UK offshore wind farm for two broadly distributed species of conservation concern: common guillemot  and harbor porpoise (Phocoena phocoena). Data were collected during boat‐based line transect surveys across a 360 km2 study area that included the Robin Rigg offshore wind farm. Surveys were conducted over 10 years across the preconstruction, construction, and operational phases of the development. Changes in guillemot and harbor porpoise abundance and distribution in response to offshore wind farm construction and operation were estimated using generalized mixed models to test for evidence of displacement. Both common guillemot and harbor porpoise were present across the Robin Rigg study area throughout all three development phases. There was a significant reduction in relative harbor porpoise abundance both within and surrounding the Robin Rigg offshore wind farm during construction, but no significant difference was detected between the preconstruction and operational phases. Relative common guillemot abundance remained similar within the Robin Rigg offshore wind farm across all development phases. Offshore wind farms have the potential to negatively affect wildlife, but further evidence regarding the magnitude of effect is needed. The empirical data presented here for two marine top predators provide a valuable addition to the evidence base, allowing future decision making to be improved by reducing the uncertainty of displacement effects and increasing the accuracy of impact assessments.Quantifying the likely effects of offshore wind farms on wildlife is fundamental before permission for development can be granted by any Determining Authority. The effects on marine top predators from displacement from important habitat are key concerns during offshore wind farm construction and operation. In this respect, we present evidence for no significant displacement from a  To date, much of the development of offshore wind energy generation has occurred in Europe where there is a binding agreement for at least a 20% share of energy consumed to come from renewable sources by 2020  and harbor porpoise (Phocoena phocoena). Both guillemot and harbor porpoise are broadly distributed in European waters and are frequently identified as important species in offshore wind farm impact assessments due to their protected status under national and international legislation  in the UK to test for evidence of displacement  of two marine top predators: common guillemot  was developed in conjunction with the Robin Rigg Management Group (RRMG) prior to construction MEMP .2.22 study area including the Robin Rigg OWF during three development phases: preconstruction, construction, and operation  unit. The time piece used for recording sightings was synchronized to the hand‐held GPS used for recording transect tracks, allowing the approximate position of each observation to be determined. Detection was undertaken by eye, and high‐quality binoculars were used to confirm species identity. Distance of each sighting from the vessel was determined using a rangefinder measuring stick  methods were followed  was scanned by a single surveyor and a strip width of 1 km was used. For each harbor porpoise sighting, time and number of individuals were recorded as a minimum. Data were collected in sea states zero to five.2.3To quantitatively compare underlying changes in relative guillemot and harbor porpoise abundance and distribution across preconstruction, construction, and operational phases of the Robin Rigg OWF, the spatial distribution of individual observations per km was modeled. Transect lines for all surveys were divided into 600 m × 600 m segments for guillemot and 1 km × 1 km segments for harbor porpoise. Guillemot and harbor porpoise observations were then assigned to these segments. Only guillemots recorded ‘on sea’ were included in the analysis; birds ‘in flight’ were excluded since it was not known whether these individuals were actively using the habitat within the study area or simply passing through  with number of guillemot per segment as the response variable. Explanatory variables included development phase and a two‐dimensional smooth of longitude and latitude which was allowed to vary by development phase . The two‐dimensional smooth of longitude and latitude acts as a proxy for any underlying spatio‐temporal covariates that are driving guillemot distribution. Surveys, and transect within survey, were incorporated as random effects to account for spatio‐temporal dependency inherent within the data. The model was used to predict relative abundance across the study area on a 0.36 km2.3.22 scale.The harbor porpoise dataset contained a large percentage of zeros and data were overdispersed when modeled using Poisson or negative binomial distributions. The model for harbor porpoise therefore consisted of a zero‐inflated Poisson generalized additive mixed effects model (ZIP GAMM) with number of harbor porpoise per segment as the response variable. Explanatory variables for the Poisson (count) part of the model included development phase and a two‐dimensional smooth of longitude and latitude which was allowed to vary by development phase . As for guillemot, the two‐dimensional smooth of longitude and latitude acts as a proxy for any underlying spatio‐temporal covariates that may be driving the distribution of the animals. The binary part of the model was used to account for additional zeros resulting from “sampling error” , “unavailability” , and “detectability” . The latter was more likely in higher sea states. As such, survey conditions were included as an explanatory variable in the binary part of the harbor porpoise model. Survey conditions were modeled as either ‘good’ (sea states of 0–2) or ‘poor’ (sea states of 3–5) in order to simplify the model and balance the covariate  Bayesian inference using JAGS Plummer,  implemen33.1Guillemots were observed across the study area during all three development phases, both within and outside the Robin Rigg OWF Figure . HoweverDevelopment phase did not predict relative guillemot abundance across the study area as a whole Table , and no 3.2Harbor porpoise were observed throughout the Robin Rigg study area during all three development phases. However, harbor porpoise were not recorded within the footprint of the Robin Rigg OWF during the construction phase Figure . Harbor Development phase did not predict overall relative harbor porpoise abundance Table . However4Improving the evidence base and reducing uncertainty surrounding the potential effects on wildlife from offshore wind energy developments will enable more informed decisions to be made as the industry expands globally  flying below the rotor‐swept area. Thus, potential collision risk is very low  is comparable to that at the Princess Amalia OWF (4.3 turbines/km2), indicating that a higher turbine density alone is unlikely to induce stronger displacement behaviors. Guillemots are only present in the Princess Amalia OWF during the winter .Robin Rigg offshore wind farm monitoring data are available free of charge via the Crown Estate Marine Data Exchange ( Click here for additional data file.


Cholangiocarcinoma is an aggressive biliary neoplasm lacking effective therapeutic agents. Immunotherapies targeting the PD-L1/PD-1 immune checkpoint have shown encouraging results in solid and hematologic cancers in clinical trials. Response to these immunomodulators is correlated with PD-L1 expression. Our goal was to characterize PD-L1 expression in intra-hepatic (iCCA) and perihilar (pCCA) cholangiocarcinomas, and to correlate our results with clinicopathological features, density of tumor-infiltrating lymphocytes (TILs) and PD-1 expression.A series of 58 iCCAs and 41 pCCAs was included in the study. PD-L1, PD-1 and CD3 expression was investigated using immunohistochemistry. Density of TILs was evaluated by immunohistochemistry using a quantitative score of CD3-stained intratumoral lymphocytes.n = 46) of the cases (31 iCCAs and 15 pCCAs). PD-L1 expression by either neoplastic or inflammatory cells was associated to high density of CD3-positive TILs . The number of PD-L1 positive inflammatory cell aggregates was higher in tumors with high PD-1 expression (p < 0.0001).PD-L1 expression by neoplastic cells was observed in 9 cases . PD-L1 positive inflammatory cell aggregates were identified in 46% (Altogether, PD-L1 in iCCA and pCCA is mainly expressed in tumors with high density of TILs. Our results suggest that CCAs with dense intratumoral lymphocytic infiltration might represent good candidates for PD-L1/PD-1 blocking agents. Cholangiocarcinomas represent a heterogeneous group of cancers derived from epithelial biliary cells. They are classified, according to their anatomic localization on the biliary tree, into intra-hepatic (iCCA), perihilar (pCCA) or distal cholangiocarcinomas . CholangRecently, clinical trials using monoclonal antibodies targeting immune checkpoint regulators, such as Programmed-death-1 (PD-1) or its major ligand, Programmed-death Ligand 1 (PD-L1), showed promising results in various hematologic and solid cancers . An objeUnder physiological conditions, PD-L1 binding to the T-cell surface receptor PD-1 induces a down-regulation of T-cell activation and signaling, as well as T-cell apoptosis, thereby suppressing auto-immunity , 12. PD-Multiple studies have sought to identify predictive markers of tumor response in order to select patients who would benefit most from immune checkpoint inhibitors , 15, 16.n = 37/89) of patients. Nevertheless, to our knowledge, specific data on the type of PD-L1 expression  and differences between intrahepatic, hilar and gallbladder cholangiocarcinoma were not yet communicated. Moreover, profile and incidence of PD-L1 expression may be different in early stage compared to advanced stage. Interestingly, 17% of responses were observed in this interim analysis [There is so far very little data on PD-L1 expression in cholangiocarcinoma , 18. Intanalysis .This prompted us to investigate PD-L1 immunohistochemical expression in a series of iCCAs and pCCAs, and to correlate our findings with the clinical and pathological features of the tumors and PD-1 expression.Ninety-nine surgically resected cholangiocarcinomas were included in the study. The median age of the patients was 62.5 years (range 24 to 85) and the sex ratio  was 2.09. Risk factors of liver disease were identified in 23 patients: hepatitis B virus infection in 8 cases, alcohol in 6, non-alcoholic steatohepatitis in 6, hepatitis C virus infection in 1, primary sclerosing cholangitis in 2, and hemochromatosis in 2. Two patients had multiple risks factors. The main clinical characteristics of the 99 patients are summarized in Table n = 58) and perihilar in 41% (n = 41). Tumor size ranged from 6 to 230 mm (mean 59.5mm) (Table n = 72) and 27% (n = 27) of cases, respectively (Table n = 41), 63% (n = 62), 55% (n = 55) and 54% (n = 36/67) of tumors, respectively. Adjacent non tumoral liver fibrosis could be analyzed in all but one case and was graded as follows: F0-F1 in 45% (n = 44), F2-F3 in 47% (n = 46) and F4 in 8% (n = 8) of cases.The tumors were intra-hepatic in 59% of cases (n = 33) and in 67% of pCCAs (n = 27/40)  Table .n = 9). PD-L1 positive tumors were intra-hepatic in 5 cases (9% of ICCAs) and perihilar in 4 cases (10% of pCCAs). The percentage of PD-L1 tumor cell expression ranged from 0 to 30%. An almost significant relationship with poor differentiation and high PD-1 expression was observed   of cases, including 31 iCCAs and 15 pCCAs  (Table PD-L1 expression by either neoplastic or inflammatory cells was associated with high density of TILs (y) Table .p < 0.0001)  . HoweverIt is unknown whether PD-L1 expression by immune cells has the same predictive value on the response to therapy as its expression by tumor cells. It is now well established that intratumoral inflammatory cells may also contribute to T cell exhaustion and anti-tumor immunity suppression through PD-L1 expression , 23. TheAltogether, this study shows that PD-L1 is mainly expressed by intratumoral inflammatory cells in both iCCAs and pCCAs, principally in tumors with dense intratumoral lymphocytic infiltration. Our results suggest that CCAs with dense intratumoral lymphocytic infiltration could be good candidates for PD-L1/PD-1 blocking agents.Patients included in the study underwent surgical resection for iCCA or pCCA at Henri Mondor Hospital, Créteil, France, between 1998 and 2015. Patients with combined hepatocellular-cholangiocarcinoma or with prior chemotherapy treatment were excluded.Age, gender and risk factors of chronic liver disease were systematically recorded. Histological slides were retrieved from the Pathology Department archives. The study was approved by the Local Ethics Committee of Saint-Louis Hospital, ‘Ile de France IV’ (IRB no. 00003835).Three pathologists  reviewed all of the available hematein-eosin-saffron stained slides for each case. Diagnosis was confirmed according to the WHO classification system . Each tuOne representative paraffin-embedded block representing the tumor area with the highest density of immune infiltration was selected. Although some representative slides included the tumor border, all immunohistochemistry stainings were evaluated strictly within the tumor, not at the tumor front.2) with greater than 100 CD3-stained TILs. Tumors with less than 10 HPFs comprising at least 100 CD3+ TILs were scored “low” and those with 10 or more were scored “high” for intratumoral lymphocytic infiltration density. CD3 staining could not be performed in one case due to block exhaustion.Density of intratumoral infiltrating lymphocytes was assessed on the representative tumor slide by immunohistochemistry using a polyclonal rabbit anti-human CD3 antibody (DAKO A0452). After examination of a training set comprising 35 tumors, CD3 expression was scored in the densest infiltrative areas within the tumor, by counting the number of high power fields (HPFs)  at pH6 (PD-1) or pH9 (PD-L1) with the primary antibodies Abcam AB52587 and Cell Signaling E1L3N, respectively. Immunodetection was performed with a biotin-conjugated secondary antibody  followed by peroxidase-labeled streptavidin and with diaminobenzidine chromogen as the substrate . Slides were processed on an automated immunostainer (Leica Bondmax) according to the manufacturer's instructions. Anti-PD-L1 and anti-PD-1 antibodies were verified and calibrated on appropriate external positive control tissues, with known constitutive positivity. Placenta was used for PD-L1 (strong syncitiotrophoblastic cell positivity) and benign lymph node tissue for PD-1 (strong positivity of follicular helper T-cells).2).PD-L1 expression was assessed in both tumor cells and tumor-infiltrating inflammatory cells. Tumor cells were considered PD-L1-positive in the presence of strong membranous staining (easily identified at X100) in at least 5% of tumor cells . InflammPD-1 was only expressed by lymphocytes, and its expression, after examination of a training set comprising 35 tumors, was assessed in 10 HPFs in intratumoral areas with the highest density of stained cells. Tumors with at least 5 HPFs comprising 20 or more PD-1 stained lymphocytes were scored “PD-1 high”, those with less were scored “PD-1 low”.p values < 0.05 were considered statistically significant.Statistical analysis was performed using Graphpad Prism 6 Software. Continuous variables and proportions were compared using Mann-Whitney and Fisher exact tests, respectively.