We previously reported that fetal plasma erythropoietin (EPO) concentrations are significantly increased in growth-restricted fetuses with abnormal umbilical artery (UA) Doppler. During hypoxia in an ovine model, the primary site of fetal EPO synthesis was switched from the kidneys to the placenta. Therefore, we designed this study to evaluate human placental EPO gene expression and the correlation to fetal serum EPO concentration in growth-restricted fetuses in a monochorionic (MC) twin model.In MC twin pairs, selective intrauterine growth restriction (sIUGR) was defined as the presence of (i) birth weight discordance of > 20% and (ii) a smaller twin with a birth weight less than the 10th percentile. Fetal UA and middle cerebral artery (MCA) Doppler were checked within 1 week before delivery. An abnormal UA Doppler was defined as persistently absent or reverse end-diastolic flow. Cerebroplacental ratio (CPR) was defined as MCA-pulsatility index (PI)/UA-PI. Fetal plasma EPO concentrations were measured in cord blood, and EPO gene expression was assayed in each twin’s placental territory. The intertwin plasma EPO ratio was calculated as the cord plasma EPO level of the smaller (or sIUGR) twin divided by the EPO concentration of the larger ) twin, and the intertwin placental EPO gene expression ratio was calculated similarly.n = 9), twins with sIUGR without UA Doppler abnormalities , and twins with sIUGR and UA Doppler abnormalities . The CPRs of smaller (sIUGR) fetuses were significantly decreased in Group 3 MC twins (p < 0.001), but not significantly different between Group 1 and Group 2. The highest fetal plasma EPO ratio and placental EPO gene expression ratio were identified in Group 3 MC twins (p < 0.001). The placental EPO gene expression ratios were significantly correlated with the fetal plasma EPO ratios .Twenty-six MC twins were analyzed, including normal twins (Group 1, This study provides evidence of increased placental EPO expression in MC twin fetuses with sIUGR and abnormal UA Doppler. Future studies are needed to confirm the similar role of placental EPO in severe IUGR singletons. In both fetuses and adults, erythropoietin (EPO) secretion is stimulated by low oxygen level . In adulMC twins share an identical genetic makeup and maternal physiology, but unequal placental sharing has been shown to result in the development of sIUGR , 8 . SIUWe have previously reported that fetal plasma EPO concentrations were significantly increased in monochorionic (MC) twins with selective intrauterine growth restriction (sIUGR) and umbilical artery Doppler abnormality . BecauseSince the contribution of the human placenta to fetal plasma EPO concentrations during hypoxia remains unclear, to fill this knowledge gap, we conducted this study to analyze the human placental EPO gene expression and correlate placental EPO expression ratios to fetal plasma EPO ratios in growth-restricted fetuses in a MC twin model.This study was conducted from October 2014 to May 2016 at Chang Gung Memorial Hospital, Linkou Medical Center, Taiwan. The study protocol was approved by the Institutional Review Board of the Chang Gung Medical Foundation (IRB #101-4803A3). The inclusion criteria required that MC twins were delivered in our hospital, mothers with MC twins provided informed consent, and cord blood and placental tissues were successfully collected. The exclusion criteria were the presence of twin-to-twin transfusion syndrome (TTTS), monoamniotic twins, twin anemia–polycythemia sequence (TAPS), and congenital, structural or genetic malformations in the fetus. Because uterine contractions may modify the fetal plasma EPO levels [The diagnosis of TTTS was based on the Quintero criteria . TAPS waPulsed-wave Doppler UA and MCA studies were performed using GE Voluson 730 Expert ultrasound machines within seven days before delivery; if more than one examination data are available then the last examination data were collected. Measurements were obtained during fetal quiescence. Means of three consecutive automated measurements were recorded for analysis for each fetus at each examination. A mid-segment of a free loop of umbilical cord was chosen as the site of measurement. Persistent UA-AREDF was recognized as abnormal UA Doppler, whereas intermittent UA-AREDF was recognized as normal UA Doppler. Measurements of the MCA indices were performed in the proximal one third of the MCA at the vessel nearest the transducer with the angle of insonation close to 0°. CPR is defined as: MCA-pulsatility index (PI)/UA-PI.In this study, sIUGR was also defined as the presence of (i) a birth weight discordance of > 20% and (ii) a smaller twin with a birth weight less than the 10th percentile , 14. BirAfter delivery of the fetus during cesarean section, the umbilical cord of each baby was doubly clamped. Before removing the placenta, surgeons on the operating table collected cord blood from the placenta by puncturing the umbilical vein. Cord blood was sent to the Department of Clinical Pathology for EPO measurements using a commercially available chemiluminescent immunometric assay . The intertwin EPO ratio was defined as the plasma EPO concentration of the sIUGR twin divided by the EPO concentration of the AGA twin.Placentas were collected immediately after delivery. After the blood was drained from the umbilical vessels, the placenta was washed with ice-cold phosphate-buffered saline to remove all blood clots and was examined for the integrity of the cotyledons and membranes. The vascular equator was defined as a border drawn in the middle of the vascular zone on the chorionic fetal surface where the communicating vessels met. The placentas were cut along the vascular equator. We collected two to three pieces of fresh placental tissue (0.5 × 0.5 × 0.5 cm) from the placental territory of each MC twin. The placental biopsies were taken from the middle layer of the placenta between the maternal and fetal surfaces. Regions with obvious calcification or infarction were avoided. The placental specimens were briefly rinsed with normal saline to remove the blood. The placental tissues were kept in RNALater reagent  at 4 °C for 24–48 h. Subsequently, the tissues were transferred to new tubes and kept in a freezer at − 70 °C for long-term storage . EPO genTotal RNA was isolated from placental tissues by using TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. Complementary DNA (cDNA) synthesis was performed as per the manufacturer’s instructions by using the SuperScript III first-strand cDNA synthesis system (Invitrogen). Quantitative real-time (RT)-PCR was performed using the SYBR Green method (Invitrogen). Placental EPO expression was analyzed in duplicate and normalized to the GAPDH values by using the following primer sequences: EPO sense: 5′-caaggaggccgagaatatca-3, EPO antisense: 5′-tccatcctcttccaggcata-3′, GAPDH sense: 5′-GGTATCGTGGAAGGACTCATGAC-3′, GAPDH antisense: 5′-ATGCCAGTGAGCTTCCCGT-3′. RT-PCR data are presented as △Ct  and the fold changes in expression . The plap values of < 0.05 were considered statistically significant.Data were analyzed using SPSS 11.0 statistical software . Intergroup comparisons of continuous variables among the three study groups were performed using one-way analysis of variance (ANOVA), followed by post hoc at least significance difference (LSD) tests for multiple comparisons. Paired Student’s t tests were used to compare continuous data within each pair. The correlations between the plasma EPO ratio and placental EPO gene expression ratio were analyzed using Pearson’s correlation coefficients. Two-tailed n = 9), normal MC twins; Group 2 (n = 9), MC twins with sIUGR but without UA Doppler abnormalities; and Group 3 (n = 8), MC twins with sIUGR and UA Doppler abnormalities.Twenty-six MC twin pairs met the inclusion criteria and were selected for this study. They were classified as Group 1  than those in Groups 1 and 2. The birth weights of both AGA and sIUGR twins were lowest in Group 3 and highest in Group 1; likely reflecting the shortest gestational age of delivery in Group 3 and longest in Group 1. The observed birth weight discordance was most severe in Group 3, mainly due to the lowest birth weights being observed in the sIUGR fetuses of this group. The maternal body mass indices (BMIs) were not significantly different among the three groups of MC twins. No significant difference was observed in the hemoglobin levels between the two fetuses in any of the groups of MC twins .The placental EPO gene expression ratios in Group 3 were significantly higher than those in Groups 1 and 2 MC twins, but no significant differences were noted between the twin pregnancies in Groups 1 and 2 (Fig. , Figs. This study reports that the placental expression of EPO in human twin pairs is associated with fetal growth restriction and abnormal UA Doppler. We did not include TTTS and TAPS in this study, so we could examine individual placental gene expression without interference by communicating blood vessels between twins. Both placental territories of MC twins share an identical genetic makeup, but the sIUGR fetus with abnormal UA Doppler findings has a smaller placental territory and receives less placental perfusion , 8. Ther1Group 3 MC twins were delivered earliest. Given the lack of significant correlations between the gestational age at delivery and plasma EPO concentrations in the sIUGR or AGA fetuses , we coulIn addition to stimulating the production of red blood cells, EPO was recently shown to have functions such as inhibition of apoptosis and stimulation of inflammatory and proangiogenic mechanisms in multiple organ systems . These nIn fetuses with chronic hypoxia, the UA-PI increase but MCA-PI decrease, reflecting a brain sparing process . The ratThe use of MC twin models is instrumental in clearly identifying the increased EPO expression levels in the less perfused placenta, because an MC twin model provides identical genetic and environmental conditions, minimizing individual, basal noises in gene expression. Thus, analysis of MC twins can reveal subtle changes that are impossible to observe in other pregnant human case–control studies.The limitations of this study are as follows. First, the histological site of EPO production in the human placenta could not be identified, although EPO mRNA has been detected at the apical tips of the fetal villi in the inner margin of the caruncle . Second,Results of this MC twin study provide human evidence of increased placentalEPO expression in sIUGR fetuses with abnormal UA Doppler. The observed increased EPO expression in this group is correlated with increased plasma EPO concentrations.


Membrane ballooning is a fundamental mechanism by which platelets contribute to thrombin generation. However, this process has not previously been described in human disease. In this study, we demonstrated the presence of ballooning procoagulant platelets free in the circulation of critically injured humans, a phenomenon which results in systemic generation of thrombin and contributes to an acute coagulopathy. The surfaces of ballooning platelets were decorated with the damage-associated molecular pattern histone H4, and exposure of healthy platelets to histone caused membrane disruption and recapitulated the phenotypic changes in injured patients. These findings provide a description of platelet ballooning contributing to human disease and identify histone release from injured tissues as a driver of the procoagulant ballooning process. Trauma hemorrhage is a leading cause of death and disability worldwide. Platelets are fundamental to primary hemostasis, but become profoundly dysfunctional in critically injured patients by an unknown mechanism, contributing to an acute coagulopathy which exacerbates bleeding and increases mortality. The objective of this study was to elucidate the mechanism of platelet dysfunction in critically injured patients. We found that circulating platelets are transformed into procoagulant balloons within minutes of injury, accompanied by the release of large numbers of activated microparticles which coat leukocytes. Ballooning platelets were decorated with histone H4, a damage-associated molecular pattern released in massive quantities after severe injury, and exposure of healthy platelets to histone H4 recapitulated the changes in platelet structure and function observed in trauma patients. This is a report of platelet ballooning in human disease and of a previously unrecognized mechanism by which platelets contribute to the innate response to tissue damage. Platelets are the primary cellular effectors of hemostasis, but become profoundly dysfunctional in critically injured patients –3. A glo9The objective of this study was to elucidate the nature of and mechanisms underpinning the phenotypic changes in platelets in critically injured patients. We examined platelets and platelet responses in blood samples taken from trauma patients immediately on arrival at the trauma center. Here we show that reduction in the ability of platelets to aggregate occurs in parallel with an increase in their procoagulant function. Using advanced image analyses, we describe the transformation of platelets into procoagulant balloons, accompanied by release of large numbers of activated microparticles which coat leukocytes. We further show that histone H4, a damage-associated molecular pattern released into the circulation as a result of tissue damage and shock , interacSI Appendix, Table S1. Using unsupervised hierarchical clustering of these variables, we identified 4 high-level clusters of patients based on variations in platelet function that had distinct clinical characteristics and outcomes . Together these data present a paradoxical situation, where critically bleeding patients lose platelet aggregatory function but maintain the ability to generate thrombin.We performed impedance aggregometry, thromboelastometry, and measured circulating prothrombin fragments in a cohort of 279 injured patients immediately on arrival in the trauma center; characteristics of these patients are reported in the outcomes A and B. outcomes C. This sSI Appendix, Table S2). We first performed transmission electron microscopy to evaluate changes in platelet morphology. Unexpectedly, we identified large numbers of balloon structures that accumulated during resuscitation and ongoing bleeding . Procoagulant platelets have also been identified as a subpopulation of activated platelets which do not bind PAC-1, a monoclonal antibody targeting the ligand-binding site on the activated conformation of integrin αIIbβ3 . Together, these data indicate a procoagulant ballooning process in trauma patients which had not been identified in human disease and occurs in proportion to the severity of injury.Platelet balloons have never been identified in human blood samples ex vivo, but have been generated in vitro and characterized as highly procoagulant . The baln αIIbβ3 . The traP < 0.001] and had a much higher proportion of PMP-coated leukocytes , molecules which are released into the extracellular space by activated, damaged, or necrotic cells after injury and which act as a signal that damage has occurred . HistoneSI Appendix, Fig. S2), consistent with the direct effect that histones are known to exert on cell membranes . Histone H4–treated platelets also produced large quantities of microparticles which retained histone H4 on their surfaces .A sustained elevation in platelet cytosolic calcium is a requirement for balloon formation , 28. In surfaces E–G and esurfaces H, suggessurfaces I, reflecThe results presented in this study demonstrate a fundamental switch in platelet behavior toward a procoagulant and proinflammatory phenotype at the expense of platelet aggregation, which occurs during traumatic hemorrhage as a direct response to tissue damage. We propose a model in which histone H4 released into the circulation by mechanically damaged or ischemic tissues exerts a direct cytotoxic effect on platelets. This interaction drives platelet ballooning, leading to the release of microparticles which interact with circulating leukocytes.Platelet balloons are thought to form at wound sites as a response to vascular injury , 28–30. 28Due to their lack of cytoskeletal architecture, balloons are delicate structures which readily disintegrate, producing large quantities of microparticles , 17. HisOur findings have important implications for the management of trauma hemorrhage and our understanding of TIC. Several authors have described a state of elevated thrombin–generating potential despite loss of procoagulant clotting factors in severely injured patients , 43. ThiIn conclusion, this study describes a dramatic phenotypic change in circulating platelets induced by histone release after major trauma. Our findings provide insights into aspects of platelet behavior previously unrecognized in trauma patients, and broaden the concept of platelet “dysfunction” during coagulopathic hemorrhage. We describe a previously undefined and fundamental component of the innate response to damage, which is manifest by the development of platelet ballooning and microparticle production. These observations have implications for the pathophysiology of trauma-induced coagulopathy and multiple-organ dysfunction, and for the future development of effective platelet therapeutics for critically bleeding patients.SI Appendix.Additional methodological details can be found in the SI Appendix, Tables S1 and S2. Healthy volunteers taking no regular medication acted as a control group (reference 07/Q0702/24).Adult trauma patients recruited into the Activation of Coagulation and Inflammation after Trauma (ACIT) study who met criteria for advanced trauma team activation at a single urban major trauma center were included in this study. Inclusion and exclusion criteria have been published previously , 5. The SI Appendix.Platelet-rich plasma was fixed in graded buffers, washed, and stored overnight in sodium cacodylate buffer. Samples were dehydrated in a graded ethanol series and then infiltrated with London Resin white resin prior to examination with a JOEL JEM-1230 microscope (JOEL USA). Further details can be found in the IIbβ3 activation, annexin V binding, and histone H4 were quantified on platelets by flow cytometry using an LSRII flow cytometer (Becton Dickinson). Platelet balloons, platelet-leukocyte interactions, and PMPs were characterized and quantified using the ImageStreamx Mk II imaging flow cyometer (Amnis). Antibody panels and gating strategies are described in the SI Appendix, Figs. S1 and S4.P-selectin (CD62P) expression, integrin α8/mL) were recalcified to 2 mM and incubated at 37 °C under stirring conditions with vehicle or Histone H4 Human, Recombinant (New England Biolabs), at the stated concentrations. Reactions were stopped by addition of 1:2 acid-citrate-dextrose . Platelets were then prepared for flow cytometry or imaging flow cytometry as described in the SI Appendix.Washed platelets (3 × 10Washed platelets were loaded with Fluo 3-AM (Biotium) for 30 min and then incubated with anti-CD42b-APC for 15 min. Platelets were then diluted 1:10 with Tyrode’s buffer with 2 mM calcium. Basal fluorescence was recorded in unstimulated platelets, and changes were quantified in real time following challenge with thrombin receptor–activating peptide 6 (TRAP) or H4 using the LSRII flow cytometer.P value of <0.05 was considered significant throughout.Hierarchical clustering analysis was performed with Morpheus software (Broad Institute). Statistical analyses were performed using Prism v6.0 (GraphPad). A 2-tailed Supplementary File


Osteosarcoma (OS) is an aggressive sarcoma, where novel treatment approaches are required. Genomic studies suggest that a subset of OS, including OS tumour cell lines (TCLs), exhibit genomic loss of heterozygosity (LOH) patterns reminiscent of BRCA1 or BRCA2 mutant tumours. This raises the possibility that PARP inhibitors (PARPi), used to treat BRCA1/2 mutant cancers, could be used to target OS. Using high-throughput drug sensitivity screening we generated chemosensitivity profiles for 79 small molecule inhibitors, including three clinical PARPi. Drug screening was performed in 88 tumour cell lines, including 18 OS TCLs. This identified known sensitivity effects in OS TCLs, such as sensitivity to FGFR inhibitors. When compared to BRCA1/2 mutant TCLs, OS TCLs, with the exception of LM7, were PARPi resistant, including those with previously determined BRCAness LoH profiles. Post-screen validation experiments confirmed PARPi sensitivity in LM7 cells as well as a defect in the ability to form nuclear RAD51 foci in response to DNA damage. LM7 provides one OS model for the study of PARPi sensitivity through a potential defect in RAD51-mediated DNA repair. The drug sensitivity dataset we generated in 88 TCLs could also serve as a resource for the study of drug sensitivity effects in OS. For those with metastatic OS at diagnosis, outcome is worse, with only 20–30% surviving for more than five years3 and is even more dismal for those with recurrent disease4. There is a paucity of early phase clinical trials available for OS patients and therefore, new or repositioned agents are needed to improve the outcome of patients. With this in mind, recent work has suggested that a significant proportion of OS, perhaps as high as 80%, exhibit genomic features reminiscent of a ‘BRCAness’ phenotype5. BRCAness describes tumours that possess histopathological and molecular characteristics similar to those found in patients with germ-line BRCA1 or BRCA2 gene defects, including a distinctive DNA repair defect arising from loss of Homologous Recombination (HR) and drug sensitivity effects associated with this HR defect, including exquisite PARP inhibitor sensitivity6. Using an analysis of whole-exome sequencing from 31 treatment naive osteosarcoma tumour samples, Kovac et al. identified large-scale genome instability signatures characteristic of BRCA1/2-deficient tumours and recurrent mutations in tumour suppressor genes associated with homologous recombination and related DNA repair processes, including FANCA BRCA2 and ATM5. A subsequent analysis of OS tumour cell lines by Engert, Kovac et al.7 suggested that two OS tumour cell line models (MG63 and ZK58) with genomic loss of heterozygosity characteristics somewhat reminiscent of BRCAness, exhibited sub-micromolar IC50 sensitivity to the clinical PARP inhibitor talazoparib . However, whether the scale of PARP inhibitor sensitivity seen in OS models resembles that in BRCA gene mutant tumour cell lines was unclear.Whilst there have been a number of successes in developing targeted approaches to carcinomas, for many patients diagnosed with hard to treat sarcomas, radical surgery and chemotherapy regimens still represent the standard of care, and survival rates have not significantly changed for some time. This is particularly true for patients diagnosed with osteosarcoma (OS), where there has been little improvement in overall survival in the past 20 years, with five year survival remaining approximately 70% for young patients with localised diseaseBRCA1 mutant tumour cell lines and to a tumour cell line model with a CRISPR-Cas9 mutagenesis-engineered PARP inhibitor resistance-causing revertant mutation in BRCA1. We find that that the majority of OS tumour cell lines, with the exception of LM7, do not exhibit profound sensitivity to PARPi that might be associated with a BRCAness phenotype.Despite the need to identify novel targeted approaches in OS and the availability of a number of relatively well characterised tumour cell lines derived from these tumours, there is a limited amount of publically available information describing the small molecule sensitivity of these models. Such information could provide the starting point for discovering novel treatment approaches for this particular sarcoma subtype. With this in mind, we describe here as a resource, a dataset detailing the chemosensitivity of 18 OS tumour cell lines, and an additional 70 tumour cell lines derived from non-OS cancer histologies, to 79 small molecule inhibitors used in the treatment of cancer or in late stage development. As part of this resource, we describe the sensitivity of OS tumour cell lines to three chemically distinct PARP inhibitors, comparing their sensitivity to BRCA1 mutation (BRCA1 p.P724fs*12) as well as a PARP inhibitor resistant SUM149-subclone, SUM149.B1*.S, that has partially restored BRCA1 function because of a CRISPR-Cas9 mutagenesis-engineered BRCA1 intragenic reversion mutation8. This BRCA1 reversion mutation restores the native open reading frame of the BRCA1 gene, recapitulates RAD51 nuclear localisation (a key molecular event in BRCA1-mediated DNA repair) and causes profound PARP inhibitor resistance8.In order to generate a resource that could be used as a starting point to investigate sensitivity to targeted agents in OS, we compiled a panel of 18 well-characterised OS tumour cell lines (TCLs) and subjected these high-throughput drug sensitivity screening, alongside 70 tumour cell lines derived from the following carcinomas: breast (34) head and neck (11), synovial sarcoma (5), cervical (4), lung (4), large intestine (3), prostate (3), soft tissue  (3), haematopoietic (1), central nervous system (CNS) (1), and hepatic (1)  gene in OS tumour cell lines has previously been shown to predict enhanced sensitivity to FGFR inhibition by the pan-FGFR inhibitor NVP-BGJ39810. To assess the ability of our screen data to identify this known chemosensitivity effect, we interrogated the screen data from OS tumour cell lines for two different FGFR inhibitors, AZ4547 and PD173074. We noted that, in general, OS TCLs exhibited greater sensitivity to AZD4547  or FGFR1 gain (HU09 and NY) were significantly more sensitive to both AZ547 and PD173074 than those OS tumour cell lines without FGFR1 copy number alterations  tumour suppressor defects in OS TCLs  effects, that are somewhat reminiscent of mutational patterns seen in tumours from cancer patients with germ-line 13, but also enhanced sensitivity to the DNA damaging agents doxorubicin, gemcitabine and camptothecin  and a BRCA1 promoter hypermethylated tumour cell line HCC3814 to display PARPi sensitivity, as did a number of synovial sarcoma tumour cell lines , as described earlier  TCLs, we extracted from the dataset the AUC Z scores for three clinical PARP inhibitors; rucaparib, olaparib and talazoparib. When we compared the sensitivity profiles of TCLs to the three different PARP inhibitors, we found these to be highly correlated Fig. . When asnce Fig. . We alsoier Fig. .Figure 2BRCA1 mutant SUM149 cells  and to display a similar level of PARPi resistance as the BRCA1 revertant SUM149 subclone  CAPAN1 pancreatic ductal carcinoma cells16 (ANOVA p = 0.3757) but significantly greater sensitivity to olaparib than BRCA1 mutant SUM149 cells (ANOVA p = 0.0119), the BRCA1 revertant SUM149 subclone, and a previously validated PARPi resistant BRCA2 revertant CAPAN1 subclone, CAPAN1.B2*.S8  and RECQL418 .The nuclear localisation of the DNA recombinase RAD51 to the site of DNA damage is a key step in the DNA repair processes mediated by BRCA1 and BRCA2 and is critical in repairing the DNA lesions caused by PARP inhibitors. This nuclear localisation of RAD51 can be estimated by microscopy-based imaging of discrete nuclear RAD51 fociWe describe here chemosensitivity profiles for a panel of OS tumour cell lines. Our intention in carrying out this work was to generate a reference dataset that could be used for a variety of applications, ranging from the selection of tumour cell models with defined drug sensitivity/resistance phenotypes for subsequent experiments, through to the identification of drug sensitivity effects associated with molecular features found in these diseases. Given the “resource” nature of the work, we have included within this report, both raw and processed data so that subsequent analyses can be facilitated. For example, as the molecular profiling of these tumour cell line models is enhanced, for example by the addition of proteomic profiles of OS tumour cell lines, the utility of the chemosensitivity profiles we describe here will no doubt be enhanced.BRCA1 mutant breast tumour cell lines, the LM7 model does display evidence of BRCAness and a PARP inhibitor sensitivity phenotype. Although this is a singular model, the LM7 cell line therefore does provide a reagent for investigating mechanisms that cause PARP inhibitor sensitivity in OS.As part of this work, we highlight how the dataset can be used to assess PARP inhibitor sensitivity in OS tumour cell lines. By comparing the PARP inhibitor sensitivity of OS tumour cell lines to the scale of sensitivity in breast tumour cell lines with BRCA1 defects and also isogenic tumour cell models with a well-established and clinically relevant mechanism of PARP inhibitor resistance, we have been able to make a comparative analysis that suggests that whilst most OS tumour cell lines do not exhibit the exquisite level of PARP inhibitor sensitivity seen in BRCA1 mutant and BRCA1 revertant clones; the synthetic lethal effects of PARP inhibitors in in vitro cell culture often require drug exposure over several cell cycles , but the five-day exposure appears sufficient here.It is important to describe the limitations of the resource we provide. Using three-dimensional tissue culture approaches would assess whether the drug sensitivity and resistance phenotypes we see here are robust in the face of the loss of an adherent surface or changes in cell morphology and organisation often more apparent in three dimensional, as opposed to two-dimensional culture systems. We also note that more prolonged drug exposures, beyond the five-day drug exposure used here, might also elicit distinct effects, especially for agents whose cell inhibitory properties are amplified by drug exposure over multiple cell cycles. Nevertheless, we did find that the five-day drug exposure used was sufficient to document differences in PARP inhibitor sensitivity between et al., 2016; Jones et al., 2017). For OS tumour cell lines, G292 (clone A141B1), MG63, CAL72, HU03N1, OSA/SJSA-1, NOS1, SAOS2, U2OS, HU09, NY and HOS were gifts from Ultan McDermott at the Wellcome Trust Sanger Institute (WTSI); LM7, OHSN, OSH25HAL (HAL) MHM, and KPD were gifts from Ola Myklebost, Univeristy of Bergen, Norway. Head and neck cancer cell line models were obtained from Susanne Gollin and Theresa Whiteside (University of Pittsburgh), Tom Carey (University of Michigan) and Hans Joenje . All cell lines were maintained as per the suppliers’ instructions. STR typing of 10 loci was performed on each cell line using the GenePrint 10 system (Promega) and used to confirm the identity of cell lines prior to storage.The origin of non-OS tumour cell lines is described in  Cell-Titre Glo (Promega) to media. After 10 minutes incubation at room temperature, Cell-Titre Glo generated luminescence was captured using a Victor X-Light plate reader. Luminescence values from each well were normalised to the median of signals from wells exposed to DMSO only  to generate surviving fractions (SF). In total, each tumour cell line was screened three times, generating triplicate SF data sets  values were calculated from dose-response survival curves  (as9) where Alow is the activity at the lowest concentration of drug used, up to the maximum tested concentration  values were used for all analyses. Proteins were identified as differentially expressed if they were associated with a p < 0.05 effect (two sided heteroscedastic t-test) and an absolute difference in log2(LFQ) greater than 3.Proteomic abundance for the osteosarcoma tumour cell lines was performed as follows: Following lysis, protein purification, and tryptic digest, peptides were separated by liquid chromatography and measured mass spectrometer. Label-free proteome quantification was performed using the MaxQuant software environmentAll processed data is present in Supplementary Tables Supplementary InformationSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6Supplementary Table 7Supplementary Table 8


Mosaic genetic variants can have major clinical impact. We systematically analyse trio exome sequence data from 4,293 probands from the DDD Study with severe developmental disorders for pathogenic postzygotic mosaicism (PZM) in the child or a clinically-unaffected parent, and use ultrahigh-depth sequencing to validate candidate mosaic variants. We observe that levels of mosaicism for small genetic variants are usually equivalent in both saliva and blood and ~3% of causative de novo mutations exhibit PZM; this is an important observation, as the sibling recurrence risk is extremely low. We identify parental PZM in 21 trios (0.5% of trios), resulting in a substantially increased sibling recurrence risk in future pregnancies. Together, these forms of mosaicism account for 40 (1%) diagnoses in our cohort. Likely child-PZM mutations occur equally on both parental haplotypes, and the penetrance of detectable mosaic pathogenic variants overall is likely to be less than half that of constitutive variants. Systematic analysis of postzygotic mosaicism (PZM) is difficult due to challenges in detecting such events. Here, Wright et al. analyse trio exome sequencing data from blood and saliva of 4,293 probands with developmental disorders from the DDD Study and estimate that >3% of causative de novo mutations result from PZM. Mutations that occur during early embryonic mitoses can result in somatic and/or germline mosaicism at appreciable levels across multiple tissues2. However, mosaicism is frequently overlooked as a source of pathogenic variation in rare monogenic diseases largely due to the challenges associated with variant detection4. Postzygotic de novo mutations (DNMs) may result in somatic mosaicism, potentially causing a less severe and/or variable phenotype compared with the equivalent constitutive mutation, or somatic and gonadal mosaicism, potentially enabling transmission of a pathogenic variant from an unaffected parent to their affected offspring  and insertion/deletions (indels)16, to large structural variants including copy number variants (CNVs)18 and chromosomal aneuploidy21. Mosaic variants have also been shown to contribute to the risk of autism spectrum disorders23. Some pathogenic mosaic variants have been shown to exhibit markedly different abundances in different tissues, due in part to differential negative and positive selective pressures in different tissues24. This differential tissue representation can be clinically relevant, particularly as some pathogenic mosaic mutations are typically absent from tissues commonly sampled for genetic testing, i.e. blood17. It is not known how common this phenomenon of differential tissue representation is for mosaic pathogenic sequence variants across different disorders.Mosaicism is a well-described biological phenomenon in which individuals harbour two or more populations of genetically distinct cells as a result of postzygotic mutationing Fig. 3–6. In aisk Fig. , with su27. Standard whole-exome sequencing (WES) typically produces data of >30X average depth, allowing detection of alleles only present in a small fraction of reads28, while ultrahigh-depth  sequencing technologies can be used to detect or confidently confirm mosaic variants present in just a tiny fraction of cells29. However, using NGS technologies, mosaic variants present in the majority of cells will often be detected by variant calling tools optimised for constitutive variation and may therefore falsely be assumed to be constitutive. This can lead to under-appreciation of mosaicism in probands and over-estimation of recurrence risks. More importantly, falsely classifying parental PZM as constitutive in both parent and child can lead to missed diagnoses due to the exclusion by bioinformatics pipelines of variants inherited from unaffected parents under the Mendelian assumptions that are typically applied in fully penetrant conditions.Despite its clinical importance, postzygotic mosaicism (PZM) can easily be missed or the variants wrongly assumed to be constitutive due to the technical challenges inherent in the detection of alleles present in only a subset of cells. Next-generation sequencing (NGS) technologies offer an opportunity to detect lower levels of mosaicism more readily than was previously possible using either capillary sequencing or microarray-based techniques21. By contrast, the study of parental and child mosaicism of pathogenic small sequence variants has been relatively piecemeal, focused primarily on specific disorders or sub-types of mosaicism30. Larger-scale analyses of likely benign sequence variants has been informative about the general properties of post-zygotic mutations10. A large-scale systematic analysis of mosaic pathogenic sequence variants is needed to determine the relative impact of different classes of mosaicism to disease.Large-scale analyses of mosaic pathogenic structural variants has been highly informative for determining the relative contributions of different classes of variants, especially in developmental disorders18. Because of the large burden of pathogenic DNMs in developmental disorders31, which account for around three-quarters of the total diagnostic yield in this cohort33, we hypothesised that an appreciable number of pathogenic mutations were likely to be mosaic.We have previously used trio-WES and SNP-array data from the Deciphering Developmental Disorders (DDD) Study to detect pathogenic mosaic structural variants in children with severe developmental disorders, and found substantial differences in copy number and loss-of -heterozygosity events between blood and saliva 34 to find pathogenic PZM—either in the affected child (child-PZM) or an unaffected parent (parent-PZM)—that might be incorrectly annotated or missed by standard NGS pipelines  diagnoses resulting from mosaic variants in our cohort.31). We designated 1000 loss-of-function (LOF) and functional DNMs in autosomal dominant DD-associated genes and X-linked dominant DD-associated genes in females as being likely pathogenic and plausibly causative, and 1490 synonymous DNMs in non-DD-associated genes as being likely benign and unrelated to the DD. To select candidate mosaic DNMs, we used a variant prioritisation strategy (see Methods) based on deviation of the proband variant allele fraction  from a binomial distribution centred around 0.5. Using this strategy, we selected 106 likely pathogenic DNMs and 143 likely benign DNMs  for ultrahigh-depth sequencing in all members of the trio. Of the likely pathogenic DNMs: 31/106 validated as mosaic DNMs in the proband (31/1000 or ~3% of likely pathogenic DNMs), of which 20 could be assayed in both blood and saliva samples; 64 validated as constitutive DNMs; three were false positives; and the validation data were uninformative in eight cases  and the strength of statistical evidence  for a deviation of VAF away from 0.5 (Table n = 4) or the presence of another more plausible genetic diagnosis (n = 3). The children with diagnoses in genes that cause well-known syndromes had phenotypes consistent with those syndromes. We were unable to determine whether the phenotypes were milder as a result of mosaicism, as knowledge of the phenotypic spectrum and individual developmental profiles of the specific disorders is limited and, together with the small numbers of affected patients, constrains drawing definitive conclusions. However, we identified a mosaic (VAF = 0.33) LOF variant in KMT2D that was considered fully diagnostic but had an intermediate methylation signature that failed to classify as either benign or pathogenic . Similar levels of mosaicism were observed in saliva and blood , only 5% of sites validated as mosaic, while 42% of sites with two or more reads validated as mosaic in the parent (n = 12) . Six of the eight low-level mosaic inherited variants were considered to be definitely or likely pathogenic following a detailed clinical evaluation; one of these six families had a second sibling with the same disorder emphasising the importance of this analysis for genetic counselling.We took two complementary approaches to evaluate parent-PZM. First, to detect low-level parental mosaicism where the variant is not called in the parental sample by standard variant calling algorithms, we started with the same list of 1000 likely pathogenic candidate DNMs described above and selected all variants with one or more alternative allele reads in a single parent for ultrahigh-depth sequencing. Of the 72 candidate variants: eight were validated as constitutive in the proband and mosaic in a parent (1% of the likely pathogenic DNMs), five maternal and three paternal; 53 variants were validated as true DNMs in the proband but absent from the parent; one variant was constitutive in both the parent and proband; six variants were false positives; and the validation data were uninformative in four cases. The number of alternate reads in the parent was highly correlated with the likelihood of validating as parent-PZM: with just a single alt read , we selected 36 candidate parental mosaic variants for validation using ultrahigh-depth sequencing. Of these: 13 variants validated as constitutive in the proband and mosaic in a parent , eight maternal and five paternal; 21 were constitutive in both the parent and proband; and the validation data were uninformative in two cases. The likelihood that a variant validated as mosaic in the parent was correlated with both the parental VAF and the strength of statistical evidence  for a deviation of VAF away from 0.5 , we identified all 18,527 rare LOF and functional variants in dominant DDG2P genes detected in the same 4293 children with DD. We then selected 10,721 constitutive heterozygous variants in the child that were also detected in just one apparently unaffected parent. Given the low prior likelihood of inherited variants being pathogenic in this cohortp-value < 0.0001 are good predictors of high-level mosaicism. For low-level mosaicism, where the variant is not detected using standard algorithms and thus we were only able to evaluate it here for parent-PZM, observing two or more reads supporting the variant allele in a parent is a good predictor parent-PZM in putative DNMs  and higher for high-level than low-level parent-PZM . We further subdivided the DNMs into classes of variant consequence  and types of genes . The correlation between enrichment of potentially damaging variants and increasing VAF is greater in classes of genes where the variants are more likely to cause monogenic developmental disorders , while the regression coefficients were significantly different for either DD-associated (p = 0.00007) or monoallelic DD-associated genes with a loss-of-function mechanism (p < 0.00001). For likely mosaic variants (VAF < 0.27) in known DD-associated genes with a monoallelic loss-of-function mechanism, the enrichment of LOF and functional variants is reduced to approximately a third that of constitutive variants (p = 0.04).The level of mosaicism detected with ultrahigh-depth sequencing was significantly higher for child-PZM than for low-level parent-PZM , the enrichment of paternal-origin mutations was significantly different (p = 0.0001) between mosaic (n = 41) and constitutive variants (n = 730). We visually inspected IGV plots for all 41 candidate mosaic DNMs with a nearby informative allele inherited from a single parent on the same read-pair. For all validated cases (n = 6), we observed the characteristic three-haplotype pattern expected for mosaic variants, and the average VAF of the inherited allele was 0.5; across all 41 variants, there is a significant difference between the VAF of the candidate mosaic DNM and the inherited variant , consistent with a 50:50 ratio expected for a postzygotic mutation arising during early embryo development, before sexual differentiation. Moreover, mosaic variants did not increase appreciably with parental age and the relationship between the number of mosaic DNMs per person and increasing parental age was not significantly different from the null , low  and minimal . The overlapping VAF distributions of PZMs in affected probands and apparently unaffected parents Fig.  indicateNMs Fig. . While t44. Detecting mosaicism is important for accurate interpretation of confirmatory biomarker assays, wherein a mosaic individual may exhibit an intermediate result that may get mis-interpreted. To exemplify this, we observed a DDD participant with a validated diagnostic child-PZM LOF variant in KMT2D who exhibited an intermediate biomarker phenotype for a published DNA methylation signature for Kabuki syndrome caused by mutations in KMT2D44 . Detecting likely mosaic variants depends on the depth of the original sequencing data, the error rate of the sequencing platform, and the metrics used to distinguish different types of variants. In principle, it should also possible to estimate the proportion of DNMs that are child-PZMs using mixture modelling on the VAF distribution of DNMs as compared to constitutive variants  and, as a consequence, only being able to detect mosaicism where the fraction was appreciably greater than the sequencing error rate. Since DeNovoGear requires >2 ALT reads to detect a variant, we estimate our power to detect child-PZM was ~75% at VAF = 0.1 and ~90% for VAF = 0.2–0.3  in which to explore tissue specificity. The correlation between the level of mosaicism in these tissues versus developmentally-relevant organs, such as the brain, is unknown and thus caution is needed when considering the correlation between observed VAF and phenotype or recurrence risk. Indeed, paternal germline mosaicism might be better evaluated using a paternal sperm sampleIn conclusion, using 4293 families with severe developmental disorders, we have shown that mosaic variants are a significant cause of rare developmental disorders, and that they can be detected from standard trio WES. The relatively low proportion of mosaic variants limited our ability to explore decreasing pathogenicity with decreasing VAF in a more quantitative manner, as well as evaluate recurrence risk empirically based on affected siblings, for which much larger cohorts will be needed. Finally, we did not have sufficient numbers of variants in any given gene to assess whether mosaic DNMs in the proband reduced the severity or expressivity of the child’s developmental phenotype relative to constitutive DNMs. There is now a need for very large-scale studies to address remaining questions and further refine our current findings regarding the relationship between variant pathogenicity and mosaicism level, further investigate tissue specificity (including gametes) and empirically estimate the recurrence risk from siblings. To empower this research endeavour, as well as increase diagnostic yields and improve clinical management, algorithms tuned to detecting mosaic pathogenic variants—particularly in an unaffected parent—should be integrated into paediatric genomics pipelines.50, and detailed developmental phenotypes for the individuals were entered by the patient’s clinician using Human Phenotype Ontology (HPO) terms51. Saliva samples for the family trio as well as blood-extracted DNA samples for the probands were collected and processed as described previously52. Trio exome sequencing was performed on either saliva or blood using Illumina HiSeq (75-base paired-end sequencing) with SureSelect baits (Agilent Human All-Exon V3 Plus and V5 Plus with custom ELID C0338371). Mapping of short-read sequences for each sequencing lanelet was carried out by the Wellcome Sanger Institute’s Human Genetics Informatics team using the Burrows-Wheeler aligner 53 backtrack algorithm with the GRCh37 1000 Genomes Project phase 2 reference . Sample- level BAM improvement was carried out using the Genome Analysis Toolkit 54 and SAMtools (version 0.1.19)53. Average read depth in the coding regions across these samples was 50X. Single nucleotide variants and indels were called using the GATK HaplotypeCaller (version 3.2.2) and GATK resource bundle (version 2.2)55, run in multi-sample calling mode using the complete data set. We used DeNovoGear 56 to detect likely DNMs from trio exome BAM files. Variants were annotated with minor allele frequencies  and the predicted consequence (using Ensembl Variant Effect Predictor49). The data are available under managed access from the European Genome-phenome Archive (Study ID EGAS00001000775), and likely diagnostic variants are available open access in DECIPHER51.The DDD Study has UK Research Ethics Committee approval . Patients with severe, undiagnosed developmental disorders and their parents were recruited and systematically phenotyped by the 24 Regional Genetics Services within the United Kingdom (UK) National Health Service and the Republic of Ireland. Clinical data  were collected using a standard restricted-term questionnaire within DECIPHER60 based on gene-specific mutation rates that account for gene length and sequence context31, and increased the stringency of called DNMs until the number of observed synonymous variants equated that expected under the null-mutation model, as published previously31. Using this high-stringency set of DNMs, we calculated the proband variant allele fraction (VAF)—defined as the read depth of the alternative allele divided by the total read depth—and calculated the binomial probability of the observed VAF given an expected VAF of 0.5 (i.e. a constitutive heterozygous variant). When identifying candidate DNMs, we applied a filtering threshold on proband VAF > 0.1 due to the difficulty of distinguishing real variants from sequencing errors at lower VAF in WES data of ~50X average depth. As a consequence, all candidate DNMs in this dataset had a proband VAF > 0.1.We generated a high-sensitivity set of 8542 rare (MAF < 0.01) candidate DNMs from 4293 WES trios. To assess the burden of mosaic SNVs and indels, we calculated the expected number of DNMs as described previouslywww.ebi.ac.uk/gene2phenotype). We then calculated the ratio of potentially damaging  DNMs to synonymous DNMs in each VAF bin and compared the linear regression coefficients for different classes of genes.To determine the parental origin of child-PZMs, we used informative SNVs inherited from a single parent on the same read-pair as the candidate DNM to determine whether the variant occurred on the maternal or paternal haplotype. To assess how variant pathogenicity varies with the level of mosaicism, we also calculated the number of different classes of DNMs in six bins of increasing proband VAF , excluding variants that fell below our stringency threshold or were on the X or Y chromosomes in males. We annotated variants with predicted loss-of-function  or functional consequences  in dominant genes known to cause developmental disorders  with a false discovery rate  of <0.2 (n = 106).Likely pathogenic DNMs: LOF and functional variants in dominant DDG2P genes  with a false discovery rate of <0.05 (n = 143).Control variants: synonymous variants in non-DDG2P genes .Candidate low-level parent-PZM: candidate likely pathogenic DNMs (see above) with any alternative allele reads in one parent were selected for validation  under two scenarios:p-value for parental VAF was calculated assuming a mean of 0.5; variants were then selected for validation using a false discovery rate (Benjamini-Hochberg) of <0.1 (n = 20);The binomial n = 10);We identified a subset of variants with a high likelihood of pathogenicity (protein-truncating variants and known pathogenic variants) and parental VAF < 0.4 (n = 6).We identified variants present in affected sib pairs with parental VAF < 0.4  inherited heterozygous LOF and functional variants in dominant DDG2P genes in 4293 probands, and annotated them with reference and alternative allele read depths across all members of the trio. We excluded variants in children with affected parents, with reads supporting the mutant allele in both parents, or with proband VAF > 0.7 or <0.3, leaving 10,721 candidates. To maximise the detection of clinically relevant parent-PZMs, we adopted three different strategies to identify 36 candidate parent-PZMs for validation from this long list of possible candidates:Candidate PZMs were selected for validation by ultrahigh-depth sequencing based primarily on statistical deviation from a VAF of 0.5. To limit the dataset to potential diploid mosaicism, we restricted our analysis of DDG2P genes to those with autosomal dominant inheritance and X-linked dominant inheritance in female probands, and to enrich our dataset for the most likely pathogenic LOF variants, further focused on genes known to cause disease via a LOF mechanism. Variants were then analysed under the following models:61 and manually confirmed in IGV44. Variant pathogenicity was assessed, for each variant, by at least two consultant clinical geneticists through a composite approach of patient assessment, variant evaluation, inheritance and clinical fit compared with previously published cases of children with pathogenic  variants in the same gene. A full list of validated mosaic variants is provided in Supplementary Data Validation of PZM candidates was performed using PCR amplification followed by Illumina MiSeq (250 bp paired-end reads) in all members of the trio, with 40 ng of genomic DNA as template and primers designed to amplify 150–250 bp products centred around site of interest. Median depth of coverage across all validations was 100,032 reads. Both saliva-extracted and blood-extracted DNA samples from the proband were assayed where available; only saliva-extracted DNA was available for parents. Variant inheritance was classified automatically using an in-house pipeline previously describedKMT2D variants was derived as described previously44 using published data (GEO: GSE97362). CpG sites with at least 10% differential methylation between KMT2D and control samples in the training set were identified and restricted to an FDR < 0.01 using a Mann-Whitney U test at each probe; the resulting CpG sites (n = 112) form the methylation signature for KMT2D. We applied this signature to 29 DDD probands profiled on the EPIC array, nine of whom had KMT2D de novo mutations of uncertain pathogenicity and 20 were age and sex matched negative controls. We assessed DNA methylation status at signature CpG sites using the Pearson correlation approach described previously44 using all 29 DDD probands and an additional 27 KMT2D variants of uncertain pathogenicity from the published test set44.To investigate the effect of mosaic mutations on a clinically relevant biomarker, we evaluated DNA methylation at ~850,000 CpG sites across the genome in a subset of individuals from the DDD study using an Illumina EPIC methylation array. A DNA methylation signature that discriminates between benign and pathogenic Supplementary InformationSupplementary Data 1Supplementary Data 2Description of Additional Supplementary FilesPeer Review File


High flow nasal therapy (HFNT) is a technique in which humidified and heated gas is delivered to the airways through the nose via small nasal prongs at flows that are higher than the rates generally applied during conventional oxygen therapy. The delivered high flow rates combine mixtures of air and oxygen and enable different inspired oxygen fractions ranging from 0.21 to 1. HFNT is increasingly used in critically ill adult patients, especially hypoxemic patients in different clinical settings.Noninvasive ventilation delivers positive pressure (end-expiratory and inspiratory pressures or continuous positive airway pressure) via different external interfaces. In contrast, HFNT produces different physiological effects that are only partially linked to the generation of expiratory positive airway pressure. HFNT and noninvasive ventilation (NIV) are interesting non-invasive supports in perioperative medicine. HFNT exhibits some advantages compared to NIV because HFNT is easier to apply and requires a lower nursing workload. Tolerance of HFNT remains a matter of intense debate, and it may be related to selected parameters. Patients receiving HFNT and their respiratory patterns should be closely monitored to avoid delays in intubation despite correct oxygenation parameters.HFNT seems to be an interesting noninvasive support in perioperative medicine. The present review provides anesthesiologists with an overview of current evidence and practical advice on the application of HFNT in perioperative medicine in adult patients. Noninvasive ventilation (NIV) delivers noninvasive continuous positive airway pressure (CPAP) or noninvasive positive end expiratory pressure (PEEP) plus an inspiratory pressure to the patient’s airway via different external interfaces , p < 0.01). However, the PaO2/FiO2 ratio was significantly greater during CPAP than during HFNT. Dyspnea improved with HFNT and CPAP but not significantly. Interestingly, when the authors evaluated patient comfort, they did not find any difference in the trials.The effects of CPAP  is a potentially life-threatening procedure in the operating room, especially in critically ill patients who require surgery. ETI may expose these patients to the risk of severe desaturation, eventually resulting in cardiac arrest and death –53.Pre-oxygenation prior ETI is a mandatory and crucial step that extends apnea time and delays the eventual desaturation. Oxygenation through a facial mask is generally recommended in patients with healthy lungs prior to general anesthesia . HoweverSimple supplemental oxygen administration prior to and/or during ETI cannot be considered a form of ventilator support. NIV reduces alveolar collapse and the likelihood of atelectasis formation, which are responsible for hypoventilation, increased perfusion-ventilation mismatch and eventual hypoxemia . NIV may2 of 1; 2) CPAP set at 7 cmH2O and FiO2 of 1; and 3) traditional facial mask at 12 L/min with an FiO2 of 1. The primary outcome was PaO2 values at defined time points during pre-oxygenation . HFNT significantly improved oxygenation during the pre-oxygenation at 5 and 7 min prior to RSI compared to traditional oxygen therapy, but no significant difference in oxygenation compared to CPAP was observed.HFNT was also evaluated in 50 patients who were at risk of difficult intubation during awake fiber-optic intubation. HFNT use was safe, well tolerated, and improved oxygenation while avoiding hypercapnia . HeinricAlthough CPAP should increase EELV by recruiting collapsed alveoli, its beneficial effect might not be associated with an increase in oxygenation . Thus, a2 levels below 90%. No patients presented adverse events related to CO2 toxicity, such as episodes of cardiac arrhythmias.Patel et al.  showed t2 of 1 was used for 4 min prior to induction and maintained until ETI  in the HFNT + NIV group compared to the NIV alone group [96 (92–99)] (p = 0.029).A recent randomized controlled trial (OPTINIV) evaluated the combination of HFNT at 60 L/min and FiOemic ARF . The priHowever, all these data were collected from ICU patients. Extrapolation to patients in the operating room is not certain.General anesthesia, surgery duration and postoperative pain may determine important modifications in the respiratory system. General anesthesia and neuromuscular paralysis induce muscle relaxation with a cranial shift of the diaphragm. This shift reduces pulmonary volumes and induces atelectasis , 68. ModOxygen administration is by far the most common therapy after extubation to correct hypoxemia. During the postoperative period, NIV use may be difficult due to the lack of a proper setting or resources  or because the patient cannot tolerate the interface . BecauseA multicenter randomized trial was performed in 527 mechanically ventilated ICU patients with a low risk of reintubation (251 post-elective or urgent surgery) . The autA further multicenter randomized non-inferiority study compared HFNT and NIV to evaluate the incidence of post-extubation ARF and reintubation in 604 high-risk patients . A totaln = 416) to NIV delivered as pressure support ventilation set at 8 cmH2O plus 4 cmH2O of PEEP, FiO2 50% administered via face mask for at least 4 h/day or HFNT (n = 414) 50 L/min with an FiO2 of 50%. HFNT was not inferior to NIV since 21% of patients in the HFNT group failed the treatment vs. 21.9% in the NIV group. ICU mortality was not significantly different between the 2 groups. Interestingly, after 24 h, nasal skin lesions were significantly more present in the NIV group.Stephan et al. performed a multicenter, randomized, non-inferiority trial (BiPOP Study) to determine whether HFNT therapy was inferior to NIV (administered as two levels of pressure - BiPAP) for the prevention or treatment of ARF after cardiothoracic surgery . PatientFutier et al. performed a multicenter randomized trial in 220 patients scheduled for major abdominal surgery (OPERA STUDY) with a moderate to high risk for developing postoperative pulmonary complications (PPCs) and compared post-extubation HFNT versus standard oxygen . The priAlthough most of the evidence on HFNT was collected from ICU studies, HFNT seems to be an interesting noninvasive support in perioperative medicine. HFNT presents some advantages over NIV. HFNT does not require patient cooperation, but it is generally better tolerated and easier to use and requires less equipment and lower nursing workload. On the other hand, since the patient does not need cooperation, surveillance by physicians must be highly reinforced to avoid delays intubation . In addiThe role of HFNT in postoperative ARF remains an open question with some important issues to be solved, such as which patient will benefit, the correct timing of treatment application and escalation. Further studies are needed to define these important issues in perioperative medicine.


Dry eye disease (DED) is the most common ocular disease and affects millions of individuals worldwide. DED encompasses a heterogeneous group of diseases that can be generally divided into two forms including aqueous-deficient and evaporative DED. Evidence suggests that these conditions arise from either failure of lacrimal gland secretion or low tear film quality. In its secondary form, DED is often associated with autoimmune diseases such as Sjögren's syndrome and rheumatoid arthritis. Current treatment strategies for DED are limited to anti-inflammatory medications that target the immune system as the source of deleterious inflammation and tissue injury. However, there is a lack of understanding of the underlying pathogenesis of DED, and subsequently, there are very few effective treatment strategies. The gap in our knowledge of the etiology of primary DED is in part because the majority of research in DED focused on secondary autoimmune causes. This review focuses on what is currently understood about the contribution of innate and adaptive immune cell populations in the pathogenesis of DED and highlights the need to continue investigating the central role of immunity driving DED. Dry eye disease (DED) is a multifactorial condition involving the ocular surface, lacrimal glands, and meibomian glands leading to abnormal tear film quantity and/or quality. Characterized by discomfort and visual disturbance, DED may lead to loss of vision caused by exposure of the ocular surface to excessive desiccant stress. Due to the high prevalence of DED worldwide, it is a critical public health issue –3.The Dry Eye Workshop (DEWS) has divided DED into two major classes: aqueous tear-deficient dry eye disease and evaporative dry eye disease. The former is often associated with autoimmune Sjögren's syndrome (ssDED) and is characterized by dysfunction of both lacrimal and salivary glands resulting in reduced tear secretion rate and/or volume. In evaporative DED, there is excessive fluid loss from the exposed ocular surface in the presence of normal lacrimal secretory function. The development of evaporative DED has also been associated with intrinsic factors such as meibomian oil deficiency, disorders of the eyelid, and low blink rate. Extrinsic factors that can also influence DED onset include vitamin A deficiency, topical drug preservatives, contact lens wearing, certain prescription drugs, and seasonal allergies .Accurate diagnosis of DED requires completion of the ocular surface disease index (OSDI) questionnaire that addresses potential risk factors as well as clinical tests to assess tear production and elimination, tear stability, and ocular surface integrity , 6. Adva+ T regulatory cells and contributes to the immune privilege of the eye [Studies have described certain immunomodulating factors involved in maintenance of the ocular surface that may be disrupted during DED. Recently, it was identified that retinoic acid, a metabolite of vitamin A, is critical for induction of Foxp3 the eye . Additio the eye –14. Stud the eye –17. Pers the eye .Therefore, inflammation associated with the eye has attracted interest from researchers worldwide in an effort to understand the immunological processes associated with the development of DED.As in many chronic diseases, sustained or dysregulated inflammation consisting of increased proinflammatory cytokine levels and infiltration of immune cells has been identified. Therefore, targeting immune cells or inflammatory mediators may have therapeutic potential. Unfortunately, little is known about the specific pathogenic cell populations in DED.In this review, unless stated otherwise, we are indicating DED as independent from autoimmune Sjögren's syndrome. We have summarized the available evidence for the role of both innate and adaptive cell populations as well as cytokines, chemokines, and their respective receptors in the pathology of DED.The following keywords were used to search the Pubmed database: dry eye disease, desiccant stress, ocular surface inflammation, and non-autoimmune. Papers published from 2000 to 2017 were reviewed. Limited focus was placed on Sjögren's syndrome-associated DED since there are already excellent reviews published , 20.The innate inflammatory response is required to eliminate potential harmful pathogens and can contribute to tissue remodeling after injury. However, if dysregulated, this type of immune response may lead to sustained inflammation resulting in compromised host organ functions. Thus, understanding the inflammatory pathways activated during DED may provide insight to potential therapeutic targets.Neutrophils are short-lived polymorphonuclear cells (PMNs) predominantly associated with frontline resistance against pathogens. In addition to their ability to phagocytize potentially harmful antigens, neutrophils can activate potent antimicrobial defense mechanisms such as the release of reactive oxygen species (ROS) and extracellular neutrophilic DNA traps (NETs) –23.Neutrophils have been detected on the ocular surface in patients with DED and are often colocalized with histone, neutrophil elastase, and extracellular DNA (eDNA) consistent with NET release. However, whether their presence is pathogenic or regulatory remains untested . A recenβ2 integrin) resulted in lower numbers of NETs under hyperosmolar conditions. An association between hyperosmolarity and NET formation has been demonstrated and suggests that there may be a potential benefit of NET inhibition during DED [Previously, it has been established that tear hyperosmolarity can trigger DED; however, whether this alters neutrophil responses is not completely understood. Tibrewal et al. reported that hyperosmolar stress, generated by increasing concentration of NaCl, induced NETosis compared to cells incubated in isoosmolar media. Furthermore, neutrophils exposed to hyperosmolar media exhibited morphological changes such as larger and rounded nuclei occupying the majority of the cell. In order to confirm the impact of hyperosmolar conditions on NET formation, the authors restored isoosmolar conditions, and subsequently, NET formation decreased. Furthermore, the addition of NET formation inhibitors  and CXCL1 chemokine in whole corneal homogenates was observed. Analysis of myeloperoxidase (MPO) activity in corneal lysates suggested a significant increase in neutrophils -1hils see . When thmediated .γ- and IL-17-producing CD4+ T cells and a significant decrease in Foxp3+ regulatory T cells  producing PMNs in the corneal limbus, lacrimal gland, and cervical lymph nodes compared to male mice. This also correlated with heightened expansion of IFN-ells see . Additioncovered .Sjögren's syndrome is an autoimmune-mediated eye pathology where one of the main identified autoantigens is the 48KDa ribonucleoprotein, also known as antigen B. Interestingly, antigen B is expressed in the nucleus and surface membrane of human neutrophils which is released when neutrophils undergo apoptosis. Whether targeting neutrophils worsens disease outcome has not been explored; however, it has been shown that antigen B activates the MAP kinase pathway and induces IL-8 output from a donor neutrophil and a neutrophil cell line .Growing evidence demonstrates that neutrophil infiltration occurs in patients with DED as well as in experimental murine models with potentially deleterious outcomes. To date, a role for early infiltrating neutrophils has been shown. However, further research is required to clarify if early versus late cellular recruitment of neutrophils can drive differential function within the tissue. Understanding more about the neutrophil phenotypes similar to other innate immune cells like macrophages is required to direct future development of therapeutic targets.γ-) or Th2- (IL-4-) dominated environments. Macrophages contribute to the spectrum of immune responses by acquiring polarized phenotypes that can be broadly classified as classically activated (M1) or alternatively activated (M2) macrophages [Macrophages are innate immune cells with a wide variety of functions ranging from recognition and clearance of pathogens to activation and polarization of adaptive immune cells. Other macrophage features include tissue repair and secretion of growth factors promoting angiogenesis . Macrophrophages , 31. The+ macrophages were found to also be positive for IL-18, which positively correlated with glandular inflammation [+ macrophages that correlated with the grade of inflammation and neovascularization [In Sjögren's syndrome, both lacrimal and salivary glands can become severely damaged. Manoussakis et al. found that when comparing patients with DED, macrophage infiltration in the periepithelial area was greater in samples from patients with Sjögren's syndrome than patients without. In contrast, dendritic cell (DC) distribution was mostly intraepithelial with consistently higher numbers in patients with Sjögren's syndrome. Interestingly, periepithelial CD68ammation . In a serization . Therefoβ, IL-6, IL-17, and CCL5) and CD4+ T cells [Due to the plastic nature of macrophages, You et al. evaluated the presence and polarity of macrophages during desiccant stress-induced DED in the cornea and conjunctival epithelium. The authors did not detect any significant changes in the number of macrophages over time in either the cornea or conjunctiva. Measurement of canonical markers of macrophage activation in conjunctival tissue demonstrated that M1-associated marker iNOS expression was increased and paralleled IL-18 high levels on day 10 postinjury; however, arginase-1 remained unchanged. This data suggests that there may be a time-dependent effect on macrophage phenotypes in DED . In line T cells . Taken t T cells .+ monocytes isolated from peripheral blood in patients with pSS had reduced ability to phagocytize apoptotic salivary gland epithelial cells compared to monocytes from healthy donors. The monocytes exhibited heightened expression of vasoactive intestinal peptide (VIP) receptor 2 (VPAC2) but not VPAC1. However, when testing monocyte response to VIP, neither phagocytic ability nor inflammatory mediator release (TNF-α) was altered in the presence of recombinant VIP [α rather than TGFβ release).Changes in circulating monocytes have also been observed during DED. In a report by Hauk et al., they showed that CD14nant VIP . Authorsγ, resulting in activation of neighboring cell populations such as macrophages and T cells. NK cells have been widely studied under a variety of pathologies ranging from infectious to autoimmune [+ TCRβ−). The percentage of conjunctival NK cells was three-fold higher in mice with DED than control mice  on APCs. In the context of nonautoimmune DED, NK cell conjunctival infiltration and disease promotion are possibly driven by IFN-γ secretion and ultimately impact APC maturation and disease outcome [Natural killer (NK) cells are a group of innate lymphocytes with cytotoxic function including secretion of membrane-disrupting granzyme and perforin largely aimed at clearing transformed and infected host cells –41. Furttoimmune –45. Althtoimmune . In a pimice see . In cont outcome .+ T cells in the ocular surface and cervical lymph nodes five days after desiccant stress exposure. Furthermore, in the absence of NK cells, authors noticed reduced expansion of CD11b+MHCII+ and CD11c+MHCII+ antigen-presenting cells (APCs). In order to confirm a central role for NK cells in Th17 expansion, Th17 cells from the spleen and cervical lymph nodes were harvested and transferred into nude mice depleted of NK cells, which later were protected against disruption of the corneal barrier. In contrast, the transfer of Th17 cells in the presence of intact NK cells developed ocular surface injury in mice. This report highlights the important role for NK cells in modulating APC activity leading to polarization of pathogenic Th17 cells involved in ocular surface inflammation [A further role for NK cells contributing to DED was described by Zhang et al. Intraepithelial NK cells were found to reside in the conjunctival tissue and are further expanded upon desiccant stress caused by scopolamine administration and airflow exposure see . Subsequammation .+ T helper cells and cytotoxic CD8+ T cells. As such, these cells are considered drivers and effectors in ongoing immune responses. Although their role is central in resistance to pathogens, both CD4+ and CD8+ T cells have been implicated in numerous autoimmune and autoinflammatory diseases. A critical role for subsets of CD4+ T cells has been described in DED as well as Sjögren's syndrome.T cells and antibody-producing B cells are the main constituent cells of the adaptive immune system. T lymphocytes can be further divided in numerous subsets including the two best characterized subpopulations CD4+ and CD8+ T cells was similar between patients with as Sjögren's syndrome DED and nonautoimmune DED, markers of T cell recruitment and activation were upregulated only in patients with nonautoimmune DED. In addition, ICAM expression was upregulated in conjunctival epithelial cells. This data suggests an important role for CD4+ T cells and integrins contributing to peripheral T cell migration and proliferation in patients with DED [The recruitment of T lymphocytes into inflamed conjunctival tissue from patients with moderate to severe DED has been characterized. Although the number of infiltrating CD4with DED .+CXCR3+ Th1 lymphocytes assayed by flow cytometry in conjunctival tissue. Thus, despite of differences in the autoimmune or nonautoimmune origin of DED, reports consistently demonstrate abundant infiltrating CD4+ T cells in ocular tissue suggesting that T cell infiltration might be a requisite to maintain the inflammatory process observed in DED.Ethnicity may also play a role in the differences between T lymphocytes in autoimmune and nonautoimmune DED. In a cohort of patients from Korea, authors reported a more severe phenotype of Sjögren's syndrome DED patients as compared to nonautoimmune DED patients . In addi+ T cell dominance over CD4+ T cells. In addition, healthy conjunctivae were found to contain a resident population of CD8+/CD69+/CD103+/CCR7− T lymphocytes. Using an array of T cell-specific surface markers, authors further characterized different T cell subsets present on the ocular surface from 52 patients with DED. Using these markers, patients were able to be clustered into different categories. Cluster 1 DED patients had high ocular redness and increased conjunctival CD8+  cells whereas cluster 2 DED patients had increased tear instability and higher proportions on conjunctival CD4+ (TEffector Memory) cells. Interestingly, most of the patients were grouped in cluster 2; however, when grouped based on DED subtype , 21% of patients did not fall into either cluster due to a unique DED phenotype with unaltered canonical DED tests (Schirmer's and break up time test) [An extensive analysis of T cells in conjunctiva samples from healthy individuals and patients with DED in Singapore was recently conducted. Bose et al. characterized T cells from healthy donors by collecting impression cytology samples  and identified a clear CD8me test) . Thus, i+ T cells from the spleen and lymph nodes of sensitized mice into nude mice resulted in a strong ocular-specific inflammatory response characterized by inflammatory foci in the cornea, conjunctiva, and lacrimal gland along with a significant decrease in the number of goblet cells and tear volume. Transfer of CD4+ T cells elicited inflammation on both the ocular surface and lacrimal gland even in the absence of airflow and desiccant stress. This study demonstrated that ocular surface-specific T cells are rapidly generated upon scopolamine sensitization and have the ability to induce DED in mice lacking T cells, suggesting the emergence of autoreactive CD4+ T cells. In addition, authors found that cotransfer of CD25+ T regulatory cells lead to a protective effect whereas this protection was lost upon depletion of Tregs during the transfer. The protective effect of T regulatory cells was also associated with a reduction in neutrophil infiltration [Murine models of DED including the scopolamine-induced desiccant stress model have been employed to study the relationship between T lymphocytes and ocular inflammation. Niederkorn et al. reported that the transfer of CD4ltration .β2, and RORγt transcription factor (expressed by committed Th17 cells) in DED patients. In confirmation, using the scopolamine-induced DED mouse model, authors found increased levels of IL-17 in tear samples from mice with DED and abundant ocular surface IL-17-producing cells. Neutralization of IL-17 led to an attenuation of DED in mice; however, the cellular source of IL-17 was not conclusively determined [A tremendous focus has been placed on the Th17 response in numerous inflammatory diseases –54. In ttermined .+ T cells rather than Th1 (IFN-γ-secreting T cells) in draining lymph nodes from animals receiving desiccant stress for 12 days. In addition, authors confirmed the presence of CCR6 chemokine receptor on the surface membrane of IL-17-secreting CD4+ T cells. The authors further quantified the expression of the CCR6 ligand CCL20 on corneal and conjunctiva tissue. Functional assays aimed to test the importance of the CCR6-CCL20 axis in DED were conducted by immune neutralization of subconjunctival-delivered blocking anti-CCL20 antibody which resulted in reduced numbers of Th17 cells in both draining lymph nodes and conjunctival samples. Moreover, this later correlated with improved clinical signs in DED mice  and decreased mRNA expression of inflammatory mediators such as IL-6, MMP3, and IFN-γ in corneal and conjunctival tissues [+CD4+ T cells in both cervical lymph nodes and the ocular surface five days after desiccant stress induction whereas CXCR3+CD4+ T cells are seen increased only on the ocular surface. The role of these CCR6 and CXCR3 chemokine receptors was tested in mice lacking either receptor. Unlike wild-type mice that exhibited compromised corneal integrity associated with increased conjunctiva infiltrating CD4+ cells and reduced goblet cell hyperplasia, mice lacking either CCR6 or CXCR3 were found to have preserved corneal integrity alongside a suppressed CD4+ cell response and unaltered goblet cell density. Additionally, IL-17 and IFN-γ responses were high in cervical lymph nodes but suppressed in the ocular surface in the absence of both chemokine receptors (CCR6 and CXCR3). To test the reduced pathogenicity attributed to T cells lacking either of the chemokine receptors, adoptive transfer experiments were carried out in T cell-deficient RAG-1 mice. Lymphocytes harvested from DED mice were transferred into naïve RAG-1 recipient animals and disease assessed. Transfer of chemokine receptor-deficient T cells inhibited the infiltration of CD4+ T cells into the conjunctival epithelium and preserved goblet cell density. When inflammatory cytokine expression was assayed, authors found that mice transferred with chemokine receptor-deficient T cells displayed a clear inhibition of virtually all the cytokines  and MMP 3 and 9 tested in corneal and conjunctival tissue [More recently, the presence of Th17 cells and their role in driving DED were addressed by means of chemically induced experimental DED. Dohlman et al. demonstrated significant expansion of IL-17-secreting CD4 tissues . The impl tissue .+ myeloid cells in experimental DED. Analysis of cornea and conjunctiva tissues showed heightened mRNA expression and protein levels of GM-CSF upon DED induction. Intracellular flow cytometry confirmed double positive staining for IL-17 and GM-CSF. To test the putative role of GM-CSF on DED pathogenesis, CD11b+ myeloid naive cells were exposed to supernatants harvested from anti-CD3-stimulated purified CD4+ T cells from DED mice with or without anti-GM-CSF antibody. Only CD11b+ cells incubated with supernatants in the presence of isotype control antibody displayed significant high levels of MHCII and Ki67 antigens indicating activation and proliferation, respectively, as compared to those cells incubated with supernatants in the presence of anti-GM-CSF-neutralizing antibodies. When anti-GM-CSF treatment was given locally to assess the in vivo impact on myeloid cells during DED development, authors reported that the treatment caused diminished infiltration of CD11b+MHCIIhi cells into the ocular surface and low numbers of Th17 cells in draining lymph nodes paralleled with attenuated clinical signs as gauged by improved corneal integrity [Recently, it was identified that GM-CSF released by Th17 cells promotes recruitment and activation of CD11bntegrity . Also, Integrity . Evidenc+CD3−) as one of the least prevalent cell populations in the ocular surface [B cells also contribute to the adaptive immune response. These cells are pivotal given their ability to secrete cytokines and as APCs; however, their key role as antibody-producing cells places them as central players in immune surveillance as well as in response to pathogen invasion. For ocular surface homeostasis, it has been reported that plasma cells are abundant in the human diffuse conjunctiva-associated lymphoid tissue (CALT) and the lacrimal drainage-associated lymphoid tissue (LDALT) and in the lacrimal glands . Plasma  surface .B cells have been studied in eye diseases from autoimmune origins including Sjögren's syndrome  and uveiIn terms of experimental DED, several reports have attempted to dissect the role of B cells. Stern et al. described that anti-kallikrein 13 antibodies arise upon desiccant stress-induced DED . Also, wMore recently, Subbarayal et al. showed that IL-17, which has been described as a highly pathogenic cytokine in DED, can also target B cells leading to their proliferation. Furthermore, neutralizing IL-17 reduced germinal center formation and the pathogenicity of transferred B cells . TherefoAlthough B cells reside in the EALT, there is no clear consensus in regard to their specific participation in the immunopathology of DED. One can speculate that discrepancies between human and mouse DED mostly rely on the stage of DED, given that most of the mouse studies have addressed this in the context of acute DED (no more than 10 days postscopolamine administration) whereas human studies are conducted in individuals being affected for longer periods of time by DED.Due to the contact between mucosal surfaces and the environment, certain immune cells are strategically positioned to rapidly respond to pathogenic threats. Early study of the ocular surface in rodents found that mast cells are present on the ocular surface and that their presence early in prenatal development may possibly contribute to the normal morphogenesis of corneal tissue , 68. HowPioneering studies on resident leukocyte populations in the conjunctiva demonstrated that eosinophils are absent in the healthy eye, but during an allergic reaction, eosinophils can infiltrate the ocular surface and contribute to a Th2 inflammatory response . EosinopAlthough basophils have been poorly studied in the settings of inflammatory ocular disorders, recently, Sugita et al. reported an important role played by early-produced type 2 cytokines ) but not IL-25 in promoting calpain-induced eye inflammation. The authors showed that ocular damage is attenuated in the absence of both IL-33 and TSLP, such phenomenon correlated with a low number of infiltrating basophils whose depletion also diminished eosinophil infiltration . Thus, aStrikingly, the relevant role of innate cells like the abovementioned granulocytes in other inflammatory ocular diseases suggests that these cells are, most likely, also involved in generating tissue injury in response to desiccating stress, however, evidence still awaits to be shown.Immune cell trafficking and activation employ soluble mediators that enable cells to infiltrate sites of tissue injury and modulate the immune response through receptor-ligand interactions. As immune system messengers, cytokines are key players in the stages of the inflammatory response. Examination of samples from patients with DED has revealed a pattern of inflammatory mediators associated with disease. Research groups have also identified a set of common mediators that can be detected in animal models of DED. A summary of these shared inflammatory mediators are shown in The eye contains numerous immune cells strategically positioned at the ocular surface with the primary goal of keeping the microenvironment tolerogenic to prevent unwanted tissue injury. In addition, resident immune cells also contribute to preserving the function of highly specialized cells involved in transduction of light stimuli into brain cells and conversion into images accomplishing the central function of the eye. Recently, reports have underscored the complex pattern of resident innate and adaptive immune cells interacting with ocular surface cells such as corneal and conjunctival epithelial cells, goblet cells, and stromal cells to maintain tear film integrity and corneal transparency which ultimately promotes ocular homeostasis. When ocular homeostasis breaks down, there is potential for the tolerogenic programs in resident cells to be altered and result in inflammatory processes that compromises both the tear film and ocular surface integrity. The triggers and mechanisms by which ocular tolerance disappears are starting to be understood, specifically in the context of DED.C. difficile infections and obesity [The challenge to come is to better define the factors behind the onset of DED. Environmental factors such as exposure to temperature controlled air, low humidity, contact lens use, extended exposure to LED-based technologies, and recently diet and excessive antibiotic use are all being considered. Growth in our knowledge of the microbiome may also become a prominent area of study. Conjunctiva-resident bacteria have been linked to modulating DED, and efforts have been made to profile conjunctiva-resident microbiota under homeostatic conditions . Startin obesity . Uncoverα therapy may have the potential to reduce DED clinical signs. Thus, identifying key cytokines driving ocular inflammation may guide the development of novel cytokine-based targeted therapies. Specifically, we recently found that the absence of macrophage migration inhibitory factor (MIF) protects against decreased tear volume and preserves tear's mucin patterns and goblet cell numbers in a scopolamine-induced DED mouse model (unpublished data). In addition, targeting nonimmune cells like goblet cells that are central in keeping tolerance through TGFβ release and mucin production opens the possibility of exploiting goblet cell expansion as another way to attenuate ocular surface inflammation.Future treatment strategies may also be developed based on existing therapeutic options for autoinflammatory diseases such as inflammatory bowel disease (IBD). In the case of IBD, application of cytokine-targeting therapies including anti-TNF-We presented numerous studies describing the complex manner for how the eye is activated upon desiccating environmental stimuli and it also has the ability to further amplify such aggressive reactions observed in immunopathogenesis of experimental and human DED. Currently, anti-inflammatory drugs and artificial tears are the most often used treatment for DED demonstrating the importance of immune response during this disease. Unfortunately, the immunological landscape is not completely understood which limits the development of new therapeutic agents. Further, DED itself is a multifactorial idiopathic disease which makes it even more complicated to treat. New therapeutics directed at counteracting DED symptoms and deleterious effects caused by sustained inflammation on ocular surface are required.In summary, inflammatory cellular infiltrate is a prominent characteristic of DED and it has been described that both innate and adaptive cells transmigrate and trigger DED. Continued examination is needed to reveal novel targets for development of effective therapeutics.